PURPOSEIntercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs).METHODSA Ca(2+) wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca(2+)](i) in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca(2+)-sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca(2+) wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence.RESULTSFlufenamic acid (FFA; 50 microM) and the connexin mimetic peptide Gap26 (300 microM), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 microM). These findings suggest that the hemichannel blockers reduce the Ca(2+) wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca(2+)-free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca(2+) removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca(2+).CONCLUSIONSHemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca(2+) wave.

The ubiquitously expressed protein Ser/Thr phosphatase‐1 isoforms PP1α, PP1β and PP1γ1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP‐tagged PP1γ1 and PP1β were predominantly targeted to the nucleoli, while PP1α showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N‐terminal residue, i.e., Gln20 for PP1α, Arg19 for PP1β and Arg20 for PP1γ1 accounts for their distinct subnuclear distribution. Our data also suggest that the N‐terminus of PP1β and PP1γ1 harbours an interaction site for one or more nucleolar interactors.

A distance continuing medical education programme was established at the Katholieke Universiteit Leuven using multipoint videoconferencing via ISDN lines at 384 kbit/s. During each academic year, 24-26 sessions were organized. During the first three years, a questionnaire was completed by 29% of the 13,489 participants. The effects of the videoconferencing on the information content and the educational effectiveness of seminars was analysed. Overall, participants were satisfied with the educational approach. Fifty-six per cent of the participants found the Pentalfa session easier to follow than conventional seminars. Participants at the peripheral sites and older participants gave higher ratings of the information content and educational effectiveness of the programme than other participants. The training of speakers and moderators was seen as useful, but most speakers could adapt themselves quite easily to the new medium.

We investigated the effect of high glucose and modulation of protein kinase C (PKC) on the intercellular propagation of Ca(2+)-waves in a rat retinal pigment epithelial cell line (RPE-J cells) in order to compare its properties with the properties previously investigated in primary LE-RPE cells. The intercellular propagation of the Ca(2+)-waves in RPE-J cells was analyzed by fluorescence imaging confocal microscopy and fluorescence recovery after photobleaching (FRAP). In control conditions the maximal normalized fluorescence in the mechanically stimulated (MS) cell and the propagation towards the neighboring RPE-J cells were similar to LE-RPE cells. As in LE-RPE cells, the propagation was reduced by the gap junction (GJ) blocker halothane, and FRAP experiments demonstrated the presence of functional GJ coupling. Similar to the effect in LE-RPE cells, the propagation of the Ca(2+)-transient was reduced by 25 mM glucose. However, unlike LE-RPE cells, the neighboring RPE-J cells presented a Ca(2+)-rise of amplitude similar to that in normal glucose levels. PKC activation with 1 microM PMA for 30 min resulted in inhibition of the Ca(2+)-wave propagation, which could be overcome by PKC downregulation as in LE-RPE cells. Cells grown for 72 h in a high glucose solution in which PKC activity was downregulated, did not develop the inhibitory effect on Ca(2+)-wave propagation that was induced by elevated glucose levels. However, the effects were not as pronounced as in LE-RPE cells. We concluded that despite marked similarities, the transduction and the modulation of intercellular propagation of the Ca(2+)-transients in RPE-J cells are not identical to the mechanisms in primary LE-RPE cells.

A distance continuing medical education programme was established at the Katholieke Universiteit Leuven using multipoint videoconferencing via ISDN lines at 384 kbit/s. During each academic year, 24-26 sessions were organized. The interactivity afforded by the videoconferencing was investigated during the first three years of the project. More than 60% of the participants rated the interactivity as good or very good. No differences were found between the sites or over the three years of the project, but female participants were more positive (66% making ratings of good or very good) in this regard than male participants (57%). Also, the effectiveness of the various types of publicity used to promote the programme was analysed. A brochure was the main source of information (62% of participants heard of the programme by this means), followed by direct mailings (27%), posters (15%), personal contact (14%) and the programme's Website (1%). The proportions of participants who heard of the programme through these different means varied with age and gender.

A distance continuing medical education programme was established at the Katholieke Universiteit Leuven using multipoint videoconferencing via ISDN lines at 384 kbit/s. During each of the first three academic years of the project, two or three periods of eight or nine sessions each were organized (a total of 74 sessions). A questionnaire was given to all participants to determine the profile of the users and the efficiency of the project. Sixty-five per cent of the participants were male. The proportion of female participants was higher at the central location than at the peripheral sites. The mean age of the participants was 37.7 years (SEM 0.3). Male participants were older than female ones. At the central location, the mean age was lower than at the peripheral locations. Nearly 90% of the participants at the peripheral sites said that they saved time by attending a videoconference session. The average time saving per participant at the peripheral locations increased from 99 min in the first year to 145 min in the third year of the project. The time saving was closely related to the distance between the peripheral location and the central location. The global cost per participant at the peripheral locations was 43.7 Euros per hour of broadcasting and the indirect estimated cost-saving was 64 Euros per hour. More than 85% of the participants at the peripheral sites stated that they would no longer travel to Leuven (the central location) for these sessions if they were to be organized in a traditional, face-to-face manner, mostly because of the time required, particularly in road travel.

A distance continuing medical education programme was established at the Katholieke Universiteit Leuven in Belgium using multipoint videoconferencing via ISDN lines at 384 kbit/s. The sessions, held in the evening, comprised oral presentations followed by interactive questions and answers, and concluded with a multi-site panel discussion. At all sites the session could be followed on two screens. The speaker/moderator or a questioner at a peripheral site, for example, was shown on the first screen and digital images were projected on the second screen. A multi-site voting system was provided. In weekly sessions between five rotating sites, a multipoint videoconference dealing with a different medical topic was presented and treated in a multidisciplinary way. During the three years of the project, 20 different peripheral sites were visited or revisited. On a five-point scale (with higher scores representing more positive evaluations), the average score for the quality of the image was 3.72 (SEM 0.01) and for sound 3.87 (SEM 0.01) (n=3743). The mean rating of the voting system was 3.76 (SEM 0.02) (n=3119). For sound, image quality and the voting system, respectively, 73%, 65% and 70% of all participants gave ratings of 'very good' or 'good'. The older participants gave slightly more positive ratings than the younger ones. Videoconferencing appears to be a. suitable alternative to face-to-face seminars.

16HBE14o- human bronchial epithelial cells predominantly express the type-3 inositol 1,4,5-trisphosphatc receptor (IP(3)R3). Besides a homogeneous localization in the cytoplasm, IP(3)R3 also displayed a Golgi-like distribution and appeared in small clusters in the perinuclear region. Addition of thapsigargin or activation of protein kinase C (PKC) during 5 hours resulted in the redistribution of these clusters to a more general cytoplasmic localization. Previously, a similar redistribution was found in A7r5 smooth-muscle cells, which express both IP(3)R1 and IP(3)R3. In the latter cells, we now show that actin interacted with IP(3)R3, while talin interacted with both IP3RI and IP(3)R3. These findings suggest a role for cytosolic and/or luminal Ca2+ and PKC in the redistribution process and a potential role of cytoskeletal proteins in the determination of IP3R localization.

In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were differentially localized. IP3R1 was predominantly localized in the perinuclear region, whereas IP3R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 μM argininevasopressin induced a redistribution of IP3R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP3R3 appeared to be unaffected. The redistribution process occurred independently of IP3R downregulation. No structural changes of the endoplasmic reticulum were observed, but SERCA-type Ca2+ pumps redistributed similarly to IP3R1. The change in IP3R1 localization induced by arginine-vasopressin could be blocked by the simultaneous addition of nocodazole or taxol and depended on Ca2+ release from intracellular stores since Ca2+-mobilizing agents such as thapsigargin and cyclopiazonic acid could induce the redistribution. Furthermore, various protein kinase C inhibitors could inhibit the redistribution of IP3R1, whereas the protein kinase C activator 1-oleoyl-2-acetyl-sn-glycerol induced the redistribution. Activation of protein kinase C also induced an outgrowth of the microtubules from the perinuclear region into the cytoplasm, similar to what was seen for the redistribution of IP3R1. Finally, blocking vesicular transport at the level of the intermediate compartment inhibited the redistribution. Taken together, these findings suggest a role for protein kinase C and microtubuli in the redistribution of IP3R1, which probably occurs via a mechanism of vesicular trafficking.

Intra- and intercellular Ca(2+)-signaling during mechanical stimulation in calf pulmonary artery endothelial cells (CPAE) was investigated with digital fluorescence microscopy. Mechanical stimulation of a CPAE cell in a Ca(2+)-containing solution revealed a rise of the free intracellular Ca(2+)-concentration ([Ca(2+)](i)) in the mechanically stimulated cell (MS) proceeding to the neighboring (NB) cells as an intercellular Ca(2+)-wave. Experiments in Ca(2+)-free solution, containing 2mM EGTA, demonstrated that a detectable [Ca(2+)](i)-transient in the MS cell is not always a requisite for intercellular communication (IC). The Ca(2+)-wave propagation was not affected by changes in membrane potential and was not mediated by voltage-dependent Ca(2+)-channels. Ca(2+)-influx through the Ni(2+)-sensitive Ca(2+)-pathway occurred in the MS as could be assessed by Mn(2+)-quenching experiments. The intra- and intercellular Ca(2+)-wave was triggered by the release of thapsigargin-sensitive intracellular Ca(2+)-stores. Phospholipase C (PLC) inhibition by U73122 reduced the Ca(2+)-amplitude of the MS cell and almost completely inhibited the IC, indicating that the Ca(2+)-release in the MS and NB cells is PLC/inositol 1,4,5-trisphosphate (IP(3)) mediated.

Intercellular Ca2+-signaling, after mechanical stimulation of calf pulmonary artery endothelial cells (CPAE), was investigated with fluorescence video imaging. Mechanical stimulation evoked an intracellular Ca2+-response in the mechanically stimulated (MS) cell, proceeding to the neighboring (NB) cells as a Ca2+-wave. The intercellular propagation of the Ca2+-wave was unaffected by the gap junction blockers halothane or heptanol. Therefore the intercellular communication (IC) pathway of the Ca2+-wave in CPAE cells does not depend on gap junctional communication but is most likely mediated by release of an extracellular mediator. Continuous unilateral flow experiments confirmed the presence of a diffusible mediator: the Ca2+-rise in upstream NB cells is significantly lower than in control experiments. After desensitization of purinergic receptors by pretreatment of CPAE cells with ATP (100mM), UTP (100μM), 2MeSATP (100μM) or ADPbS (100μM), the propagation of the intercellular Ca2+-wave upon mechanical stimulation was significantly inhibited. Also suramin (200 and 400μM), a non-specific purinergic receptor blocker, reduced the IC. Application of the nucleotidase apyrase VI (10U/ml), which has a high ATPase/ADPase ratio, enhanced Ca2+-signaling and IC. In contrast, apyrase VII (10U/ml), which has a high ADPase/ATPase ratio, significantly depressed the propagation of the intercellular Ca2+-wave upon mechanical stimulation. Our experiments therefore demonstrate that the IC, evoked by a mechanical stimulus of CPAE cells, is mediated via release of nucleotides in the extracellular space. The data indicate that the diffusible messenger, responsible for the propagation of a Ca2+-wave, is mainly ADP or a combination of ADP/ATP.

. Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions.

Experiments using confocal laser microscopy on the rat osteosarcoma cell line (ROS 17/2.8) indicate that mechanical stimulation elicits pronounced [Ca2+]itransients in the MS (mechanically stimulated) cell, which then propagate to the NB (neighbouring) cells. Experiments with Ca2+-free solutions or gadolinium suggest that Ca2+-influx through stretch-sensitive channels is required. When intracellular stores are depleted with thapsigargin, mechanical stimulation was able to evoke a Ca2+transient of reduced amplitude that disappeared entirely after subsequent blocking of Ca2+-influx. Heptanol inhibited intercellular propagation of the Ca2+transient, demonstrating the involvement of gap junctions in the propagation of the Ca2+transient in ROS cells. PKC activation has only a small inhibitory effect, while inhibition of PKC or tyrosine kinase was ineffective. PKA activation reduced the amplitude of the [Ca2+]i-rise in NB cells, and decreased the percentage of responsive cells. Cells grown in 50mM glucose for 72h presented only a very limited decrease of the Ca2+-rise during mechanical stimulation in the MS and NB cells compared to control conditions. PKC downregulation in high glucose did not modulate this effect. The results of our experiments indicate that PKC or sustained high glucose concentrations do not affect gap junctional communication in ROS cells, while activation of PKA has an inhibitory effect. This might indicate that osteoblastic dysfunction in diabetes could be directly related to the high glucose concentrations and not to inhibition of the intercellular communication.

Macrophage infiltration into the subendothelial space at lesion prone sites is the primary event in atherogenesis. Inhibition of macrophage homing might therefore prevent atherosclerosis. Since HDL levels are inversely correlated with cardiovascular risk, their effect on macrophage homing was assessed in apoE-deficient (apoE(-/-)) mice. Overexpression of human apolipoprotein AI in apoE(-/-) mice increased HDL levels 3-fold and reduced macrophage accumulation in an established assay of leukocyte homing to aortic root endothelium 3.2-fold (P<0.005). This was due to reduced in vivo beta VLDL oxidation, reduced beta VLDL triggered endothelial cytosolic Ca2+ signaling through PAF-like bioactivity, lower ICAM-1 and VCAM-1 expression, and diminished ex vivo leukocyte adhesion. Adenoviral gene transfer of human PAF-acetylhydrolase (PAF-AH) in apoE(-/-) mice increased PAF-AH activity 1.5-fold (P<0.001), reduced beta VLDL-induced ex vivo macrophage adhesion 3.5-fold (P<0.01), and reduced in vivo macrophage homing 2.6-fold (P<0.02). These inhibitory effects were observed in the absence of increased HDL cholesterol levels. In conclusion, HDL reduces macrophage homing to endothelium by reducing oxidative stress via its associated PAF-AH activity. This protective mechanism is independent of the function of HDL as cholesterol acceptor. Modulation of lipoprotein oxidation by PAF-AH may prevent leukocyte recruitment to the vessel wall, a key feature in atherogenesis.

In this chapter, current topics in the year 1996 about smooth muscle with regards to Ca2+ storage, Ca2+ release and reuptake, and Ca2+ regulation of contraction are discussed. Ca2+ is mobilized from the endoplasmic reticulum, the physiologically important Ca2+ reservoir in vascular smooth muscle, through two Ca2+-release channels: inositol trisphosphate and the ryanodine receptor. The characteristics, function, and control of these two receptors are summarized. Furthermore, evidence for a role of the nucleus as a potential Ca2+ storage site is presented and the role of mitochondria and Ca2+ extrusion systems are discussed. Also, an overview of the current understanding of the mechanism of contraction and relaxation of smooth muscle is given and the modulation of the Ca2+ sensitivity of the force is discussed.