Sustained conduction of vasomotor responses in rat mesenteric arteries in a two-compartment in vitro set-up.

AIM: Conduction of vasomotor responses may contribute to long-term regulation of resistance artery function and structure. Most previous studies have addressed conduction of vasoactivity only during very brief stimulations. We developed a novel set-up that allows the local pharmacological stimulation of arteries in vitro for extended periods of time and studied the conduction of vasomotor responses in rat mesenteric arteries under those conditions. METHODS: The new in vitro set-up was based on the pressure myograph. The superfusion chamber was divided halfway along the vessel into two compartments, allowing an independent superfusion of the arterial segment in each compartment. Local and remote cumulative concentration-response curves were obtained for a range of vasoactive agents. Additional experiments were performed with the gap junction inhibitor 18ß-glycyrrhetinic acid and in absence of the endothelium. RESULTS: Phenylephrine-induced constriction and acetylcholine-induced dilation were conducted over a measured distance up to 2.84 mm, and this conduction was maintained for 5 minutes. Conduction of acetylcholine-induced dilation was inhibited by 18ß-glycyrrhetinic acid, and conduction of phenylephrine-induced constriction was abolished in absence of the endothelium. Constriction in response to high K+ was not conducted. Absence of remote stimulation dampened the local response to phenylephrine. CONCLUSION: This study demonstrates maintained conduction of vasoactive responses to physiological agonists in rat mesenteric small arteries likely via gap junctions and endothelial cells, providing a possible mechanism for the sustained functional and structural control of arterial networks.

On the autofluorescence of fingermarks.

The autofluorescence of fingermarks is used for their detection. The components responsible for this autofluorescence are largely unknown. Thin layer chromatography and fluorescence spectroscopy were used to identify autofluorescent components and evaluate their forensic value. Based on our results, tryptophan is hypothesized to be a major contributor to the autofluorescence when part of peptides or proteins, id est, not in its free form. Part of the autofluorescence could be assigned to a kynurenine derivative. Pheophorbide A, a metabolite of chlorophyll, is inferred as a red fluorescent fingermark component. Chlorophyll is a plant pigment which implies that dietary information can potentially be retrieved from fingermarks.

A novel quantitative method for evaluating diffuse in-stent narrowing at follow-up angiography.

A new quantitative parameter, diffuse index (DI), was proposed to evaluate objectively whether in-stent restenosis is diffuse or focal in nature. A total of 343 patients (346 lesions) with Wiktor-GX, AVE MS-II, or JOMED stents were evaluated at follow-up angiography. According to the QCA-CMS definition, lesion length is derived from the 100% reference diameter function (RDF). By moving the RDF downward, the lesion length, LL(x), at each percentage x of the RDF can be calculated. We have defined the DI by the ratio of this calculated length LL(x) and the total stent length, SL, in other words, DI = [LL(x)/SL]. The percentage plaque area (% PA) was calculated by dividing the plaque area by the sum of the plaque area and luminal area within the stent. An excellent correlation was found between the DI at 88% RDF and the % PA in all three stents (r > 0.88). The individual correlation curves were nearly identical, independent of the type of stent. Furthermore, based on the overall data, the combination of a DI > 0.8 and % PA > 30% correlated with a high incidence of subsequent major adverse cardiac events (13/25 = 52%). From these data, it can be concluded that the diffuse index is a new objective quantitative parameter to describe whether in-stent restenosis is of focal or diffuse nature.

Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralisation of endosomal pH nor osmotic swelling of endosomes.

The anti-malaria drug primaquine is a weak base which accumulates in endosomes in a protonated form and consequently neutralises the endosomal pH. Bafilomycin A1 prevents endosome acidification by inhibiting the vacuolar proton pump. Although both agents neutralise the endosomal pH, only primaquine has a strong inhibitory effect on recycling of endocytosed proteins to the plasma membrane (Van Weert et al. (1995), J. Cell Biol. 130, 821-834). This suggests that primaquine interferes with a parameter, other than endosomal pH, that is essential for membrane recycling. In the presence of 0.3 mM primaquine, endocytosed transferrin-receptors accumulated intracellularly, but not in the additional presence of bafilomycin A1. Thus, at relative low concentrations proton pump-driven accumulation of primaquine in endosomes was required to inhibit membrane recycling, suggesting that the target of primaquine is associated with endosomes. The inhibitory effect of 1 mM primaquine on transferrin receptor recycling was not reversed by the additional presence of bafilomycin A1, indicating that osmotic swelling of endosomes due to accumulation of protonated primaquine could also not explain its effect. To study endosome swelling morphologically, we introduce a novel technique for fluorescent labelling of endosomes involving HRP-catalysed biotinylation. In the presence of 0.2 mM primaquine endosomal vacuoles with diameters up to 2 microm were observed. Endosome swelling was not observed when in addition to primaquine also bafilomycin A1 was present, supporting the notion that vacuolar proton pump activity lowers the dose response for primaquine. Factors that are crucial for membrane recycling and may be affected by primaquine are discussed.

Heterogeneous behavior of cells with respect to induction of retrograde transport from the trans-Golgi network to the Golgi upon inhibition of the vacuolar proton pump.

We investigated whether the vacuolar proton pump is required for proper vesicular trafficking through the trans-Golgi network. Previously, we provided evidence that endocytic transport to early and late endosomes, as well as recycling from endosomes to the plasma membrane, but not transport to lysosomes, continued in the presence of bafilomycin A1, a specific inhibitor of the vacuolar proton pump (Van Weert et al., J. Cell Biol. 130, 821-834 (1995)). Following endocytosis, recycling of cell surface glycoproteins occurs directly from endosomes to the plasma membrane, but also involves trafficking via the trans-Golgi network. We now used transferrin conjugated to horseradish peroxidase (Tf/HRP) to label and monitor the itinerary of the transferrin receptor using electron microscopy. In the presence of bafilomycin A1, transport of Tf/HRP to the trans-Golgi network continued. However, in a limited percentage of cells bafilomycin A1 induced transport of Tf/HRP into the Golgi stack. Retrograde transport into the Golgi stack was analyzed further in cells that were additionally treated with Brefeldin A, a drug that induces a redistribution of Golgi, but not trans-Golgi network markers, to the endoplasmic reticulum and the nuclear envelope. Sequential addition of bafilomycin A1 and Brefeldin A to exponentially growing A431 cells resulted in heavy Tf/HRP-labeling of the endoplasmic reticulum and the nuclear envelope in about 3% of the cells. No Tf/HRP was detected in endoplasmic reticulum or nuclear envelope of the other cells. the same observation was made for HepG2, CHO, and HeLa cells or when HRP was used as a fluid phase-endocytosed marker. Experiments performed with cell cycle-synchronized A431 cells revealed that retrograde transport was not linked to any specific phase of the cell cycle. The presence of epidermal growth factor, however, increased the amount of positive nuclear envelopes in A431 cells to 8%. In conclusion, our results show that inhibition of the vacuolar proton pump by bafilomycin A1 induces retrograde transport from the trans-Golgi network into the Golgi, and indicate that clonal cell lines display heterogeneity in this respect.

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.

Characterization of ecto-ATPase on human blood cells. A physiological role in platelet aggregation?

Ecto-ATPase (EC 3.6.1.15) is a plasma membrane-bound enzyme which degrades extracellular triphosphate nucleotides. Although its physiological function is still unclear, the enzyme obscures the study of P2 purinoceptors (i.e. receptors for ATP and other di- and triphosphate nucleotides), since it is capable of metabolizing the pharmacological ligands, such as ATP, for these receptors. We characterized the ecto-ATPase activity on human blood cells with a [gamma 32P]ATP assay and HPLC measurements. We also determined whether ecto-ATPase activity could affect the anti-aggregatory role of ATP in whole human blood. The Km for ATP of the ecto-ATPase on human blood cells was 8.5 +/- 2.3 microM and the maximum degradation rate, at 37 degrees, was 2.7 +/- 1.1 nmol ATP/(min x mL whole blood). In whole blood the major part of ATP was broken down by the blood cells, predominantly by the leukocytes. ATP and UTP were broken down equally well, mainly yielding the corresponding di- and monophosphates. In search of inhibitors for the ecto-ATPase, we studied several analogs of ATP. 8-Bromo-ATP as well as 2'- and 3'-deoxy-ATP were substrates for the enzyme. In contrast, modification of the phosphate side chain yielded inhibitors. Subsequently, a possible role of the ecto-ATPase in platelet aggregation was verified. To assess the role of the plasma membrane-bound enzyme, platelet aggregation was determined in whole blood instead of platelet-rich plasma. In the presence of ATP alone, an antagonist of ADP-induced platelet aggregation, some aggregation was still observed. As breakdown of ATP by the ecto-ATPase leads to gradual formation of ADP, as mentioned above, we compared the effects of a stepwise versus bolus addition of ADP. Subsequent dosing of ADP (1.5, 2.5, 5 and 10 microM) resulted in platelet aggregation but to a much smaller extent, at most approximately 60%, compared to the amount of platelet aggregation obtained with a bolus addition of ADP (10 microM). In conclusion, human blood cells possess a high affinity ecto-ATPase which degrades ATP as well as ATP analogs with modified base and ribose moieties. ATP analogs with a modified phosphate chain are inhibitors of the ecto-ATPase. A direct role of the ecto-ATPase activity on platelet aggregation is probably small, as degradation of ATP to ADP proceeds slowly and cumulative addition of ADP to platelets in whole blood results in a modest amount of aggregation.(ABSTRACT TRUNCATED AT 400 WORDS)

[3H]R75231--a new radioligand for the nitrobenzylthioinosine sensitive nucleoside transport proteins. Characterization of (+/-)-[3H]R75231 binding to calf lung membranes, stereospecificity of its two stereoisomers, and comparison with [3H]nitrobenzylthioinosine binding.

The tritiated analogue of R75231 ((+-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis (4-fluorophenyl)pentyl]-1-piperazineacetamide) has been examined as a new radioligand for (nitrobenzylthioinosine sensitive) nucleoside transport proteins. [3H]R75231 was prepared in two steps from R69064 ((+-)-4-[5,5-bis[4-fluorophenyl)-4-pentenyl]-2-piperazinecarboxamide+ ++ dihydrochloride) with a specific activity of 0.23 TBq/mmol (6.3 Ci/mmol). [3H]R75231 bound in a pseudo-irreversible and saturable manner to a membrane preparation of calf lung tissue. The new radioligand displayed high affinity (KD = 0.32 +/- 0.06 nmol/l at 25 degrees C) and capacity (Bmax = 6.1 +/- 0.3 pmol/mg protein). Specific [3H]R75231 binding could be fully displaced by both structural analogues and reference inhibitors such as dipyridamole, NBI, dilazep and hexobendine, as well as by various nucleosides. The two stereoisomers of R75231, R88016 ((+)-R75231) and R88021 ((-)-R-75231), potently displaced specific [3H]R75231 and [3H]NBI binding, R88021 being 30-fold more active than R88016. Pseudo-Hill coefficients derived from the shape of all the [3H]R75231 displacement curves were approximately unity. In contrast, R75231 and most of its analogues displaced specific [3H]NBI binding with pseudo-Hill coefficients consistently larger than unity under identical experimental conditions. This latter finding is suggestive for the existence of two distinct binding sites for the two radioligands, which may or may not overlap to some extent.