Visual detection of transport-P in peptidergic neurones.

1. Hypothalamic peptidergic neurones possess an uptake process for amines (transport-P), for which prazosin is a substrate. It is characterized by a paradoxical increase in the accumulation of [3H]-prazosin when the concentration of unlabelled prazosin is increased above 10(-7) M. This increase is due to activation of a proton-dependent, vacuolar-type ATPase-linked pump that is blocked by tricyclic antidepressants. This study utilized a fluorescence method to detect amine uptake in individual cells. 2. Prazosin is fluorescent but most of its emission spectrum is in the ultraviolet range. We therefore used an analogue of prazosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green part of the visible spectrum. 3. BODIPY FL prazosin accumulated in immortalised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL prazosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for prazosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL prazosin were accumulated by the same uptake process. 4. BODIPY FL prazosin accumulated in a granular distribution, which is compatible with storage in intracellular vesicles. 5. Hypothalamic cells from foetal rats in primary culture also accumulated BODIPY FL prazosin by a desipramine-sensitive process. Uptake was predominantly in neurones and glial cells did not accumulate the amine. 6. Fluorescent detection provides visual evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain.

CD44 expression in benign and malignant nevomelanocytic lesions.

CD44 is an integral membrane glycoprotein that is a principal receptor for hyaluronan and plays a role in cell-extracellular matrix interactions. Recent studies of melanomas in mouse models have suggested that increased CD44 expression by these tumors may relate to metastatic potential. Immunohistochemical expression of CD44 (standard [s] and variant [v6]) in benign and malignant nevomelanocytic lesions was assessed in formalin-fixed, paraffin-embedded tissue and was correlated with histological parameters and prognostic factors. Cases included benign nevi (three junctional, four compound, five intradermal, five blue, six Spitz, one deep penetrating), architecturally disordered (dysplastic) nevi (three, and primary (22) and metastatic melanomas (eight). All of the benign lesions showed diffuse and essentially uniform membrane staining of CD44s in nevomelanocytic cells, regardless of lesion size, depth, or extent of dermal involvement. In contrast, semiquantitative analysis (0 to 3+) of the primary melanomas showed heterogeneous and decreased staining of CD44s, which inversely correlated with lesion size (-0.569) and depth of invasion (-0.622 and -0.617 for Breslow's depth and Clark's level, respectively). These results were significant at P < .05. CD44s expression in metastases paralleled that of their respective primaries. None of the benign nevomelanocytic lesions showed CD44v6 staining. In contrast, all of the malignant nevomelanocytic lesions showed cytoplasmic staining of the tumor cells. Pretreatment with chondroitinase did not alter CD44s staining. CD44s expression by immunohistochemical determination is uniform in benign nevomelanocytic lesions. Malignant melanomas show decreased, heterogeneous staining that inversely correlates with increasing size, depth, and level of invasion. CD44 expression may be a prognostic indicator in malignant melanomas. Tumor staining with anti-chondroitin sulfate monoclonal antibodies suggests that CD44s may be expressed as a chondroitin sulfate proteoglycan in primary melanomas.

Cell surface chondroitin sulfate proteoglycans in tumor cell adhesion, motility and invasion.

Tumor cell invasion and metastasis is highly dependent on dynamic changes in the adhesion and migration of transformed and malignant cells. As with normal cell adhesion, the adhesion of tumor cells influences their cytoskeletal organization, activation of signal transduction pathways within the cell, and nuclear events leading to changes in mRNA transcription and protein synthesis. Furthermore, as tumor cells invade the circulation, they adhere to activated endothelial cells at sites within the vasculature during arrest and extravasation. Studies in the area of tumor cell adhesion and migration have demonstrated that the recognition of extracellular matrix ligands, or adhesion promoting ligands expressed on neighboring cells (i.e. counter-receptors), involves complex molecular recognition mechanisms. The complexity arises, in part, from the multiple recognition sites that are present within adhesion promoting ligands. Some of these structures within ECM components act by binding integrins, whereas others bind additional receptors such as cell surface proteoglycans. In this sense, adhesion promoting ligands may be considered as informational arrays, that function to modulate cell phenotype by engaging specific combinations of adhesion receptors on the cell surface. Understanding the mechanism(s) by which these receptor 'cluster' modify cell adhesion, motility and growth may lead to novel therapeutic strategies to control tumor cell invasion and metastasis formation. This review will highlight the role that cell surface chondroitin sulfate proteoglycans may play in modulating tumor cell adhesion, migration and invasion, with an emphasis on the relationship between cell surface chondroitin sulfate proteoglycans and integrins.

CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix.

Microvascular endothelial cell invasion into the fibrin provisional matrix is an integral component of angiogenesis during wound repair. Cell surface receptors which interact with extracellular matrix proteins participate in cell migration and invasion. Malignant cells use CD44-related chondroitin sulfate proteoglycan (CSPG) as a matrix receptor to mediate migration and invasion. In this study, we examine whether cell surface CSPG can mediate similar events in nonmalignant wound microvascular endothelial cells or whether use of CSPG for migration and invasion is a property largely restricted to malignant cells. After inhibiting CSPG synthesis with p-nitrophenyl beta-d xylopyranoside (beta-d xyloside), wound microvascular endothelial cells were capable of attaching and spreading on the surface of a fibrin gel; however, their ability to invade the fibrin matrix was virtually eliminated. To begin to examine the mechanism by which endothelial cells use CSPG to invade fibrin matrices, cell adhesion and migration on fibrinogen was examined. Endothelial cell adhesion and migration on fibrinogen were inhibited by both beta-d xyloside and after cleavage of chondroitin sulfate from the core protein by chondroitinase ABC. We have determined that wound microvascular endothelial cells express the majority of their proteoglycan as CSPG and that the CSPG core protein is immunologically related to CD44. PCR studies show that these cells express both the "standard" (CD44H) isoform and an isoform containing the variably spliced exon V3. In addition, anti-CD44 antibody blocks endothelial cell migration on fibrinogen. Affinity chromatography studies reveal that partially purified microvascular endothelial cell CSPG binds fibrinogen. These findings suggest that CD44-related CSPG, a molecule implicated in the invasive behavior of tumor cells, is capable of binding fibrinogen/fibrin, thereby mediating endothelial cell migration and invasion into the fibrin provisional matrix during wound repair.

CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion, migration, and invasion in vivo.

A peptide model of basement membrane collagen alpha 1 (IV) 531-543 binds the alpha 3 beta 1 integrin.

Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning alpha 1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B. (1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the alpha 1(IV)-531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the alpha 1(IV)531-543 chemically synthesized peptide was inhibited by a monoclonal antibody against the alpha 3 integrin subunit, and to a lesser extent by monoclonal antibodies against the beta 1 and alpha 2 integrin subunits. An anti-alpha 5 monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an alpha 1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the alpha 3 or beta 1 integrin subunit, proteins corresponding in molecular weight to alpha 3 and beta 1 integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the alpha 2 or alpha 5 integrin subunits. Thus, the alpha 3 beta 1 integrin from two tumor cell types has been shown to bind directly to the alpha 1 (IV)531-543 peptide. The alpha 1(IV)531-543 peptide is the first collagen-like sequence that has been shown to bind the alpha 3 beta 1 integrin.

Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy.

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation.

Interaction of influenza hemagglutinin amino-terminal peptide with phospholipid vesicles: a fluorescence study.

We have studied tryptophan fluorescence from a 20-residue synthetic peptide corresponding to the amino terminal of the HA2 subunit of the influenza virus hemagglutinin protein, a putative "fusion" peptide. Decay-associated spectra have been obtained at pH 7.4 and at pH 5 (the optimal pH for influenza virus fusion) in the presence and absence of liposomes. We demonstrate that a blue shift in the total steady-state fluorescence spectrum upon binding to liposomes is due to a movement in characteristic emission wavelength and increased lifetime of one of the resolved spectral components. In contrast, a further shift after lowering the pH is the product of a redistribution in the relative amplitudes of spectral components. Also, each decay component is quenched by spin-labels or anthroxyl groups normally located within the hydrocarbon interior of the membranes. Calculations are presented leading to an estimate of the distance of the tryptophan residue from the bilayer center, suggesting that the tryptophan residues are at or near the hydrocarbon-polar interface. No gross positional change was detected between pH values. Rotational depolarization is shown to be retarded by liposome binding, more so at low pH.

Nanosecond time-resolved fluorescence kinetic studies of the 5,5'-dithiobis(2-nitrobenzoic acid) reaction with enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498). Previous studies revealed two major lifetimes and spectra, suggesting distinct environments for tryptophan. In this paper, we examine the dynamic quenching of tryptophanyl fluorescence that occurs when an energy transfer acceptor, thio-2-nitrobenzoic acid (TNB), is covalently attached to the sulfhydryl groups. More precisely, we have traced the recovery of nativelike fluorescence lifetime components (and the concomitant loss of "reduced lifetime" amplitudes) that accompanies TNB release. The course of lifetime changes seen when a reducing reagent removes the quencher may be sensitive to a variety of effects, including different SH affinities, different proximities to Trp, changing availability for dimerization, or conformational changes. The prospective value of separating each lifetime component from the mixture is illustrated.