Bernard Himpens

Free‐calcium and force transients during depolarization and pharmacomechanical coupling in guinea‐pig smooth muscle.

1. Fura2 was loaded by permeation and hydrolysis of the acetoxymethyl ester into smooth muscle cells of intact thin sheets of the longitudinal layer of the small intestine of the guinea‐pig, to record Ca2+ transients during contraction. 2. Cytoplasmic Ca2+ ([Ca2+]i) was monitored by computing the ratio of the fluorescence signal excited at 340 and 380 nm wavelengths. The dye loading and the exposure to UV light required for the experiments had no significant effect on the contractile parameters observed. 3. Spontaneous, rhythmic increases in [Ca2+]i were often observed, preceding the onset of force. Removal of extracellular Ca2+ caused a very transient increase in [Ca2+]i accompanied by a phasic force transient; this was followed by a decline in [Ca2+]i and tension below control levels. Elevated Ca2+ from 1.2 to 15 mM also caused a fall in [Ca2+]i and a relaxation of basal tension. 4. Elevation of [K+]o increased [Ca2+]i. Graded concentrations of K+ caused graded changes in both fluorescence ratio and tension. 5. Carbachol evoked a transient increase in [Ca2+]i and contraction. Thereafter, in spite of the continued presence of the drug, both signals declined, presumably as the result of cholinergic desensitization. The initial phasic force response to carbachol was usually followed by an after‐contraction, that was only occasionally accompanied by a similar (small) secondary rise in the fluorescence signal. 6. In depolarized smooth muscle, both in the presence and in the absence of extracellular Ca2+, carbachol induced a transient increase in [Ca2+]i, indicating that Ca2+ release from intracellular stores is a major mechanism of pharmacomechanical coupling. 7. In some preparations an applied stretch caused, after a few seconds, a rise in [Ca2+]i and force development.

HDL-associated PAF-AH reduces endothelial adhesiveness in apoE−/− mice

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 βVLDL oxidation, reduced βVLDL 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 β 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.—Theilmeier, G., De Geest, B., Van Veldhoven, P. P., Stengel, D., Michiels, C., Lox, M., Landeloos, M., Chapman, M. J., Ninio, E., Collen, D., Himpens, B., Holvoet, P. HDL‐associated PAF‐AH reduces endothelial adhesiveness in apoE−/− mice. FASEB J. 14, 2032–2039 (2000)

Pannexins, distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates. About 10 years ago, however, a new GJ‐forming protein family related to invertebrate innexins (Inxs) was discovered in vertebrates, and named the pannexin (Panx) family. Panxs, which are structurally similar to Cxs, but evolutionarily distinct, have been shown to be co‐expressed with Cxs in vertebrates. Both protein families show distinct properties and have their own particular function. Identification of the mechanisms that control Panx channel gating is a major challenge for future work. In this review, we focus on the specific properties and role of Panxs in normal and pathological conditions.

Calcium ion homeostasis in smooth muscle

Ca2+ plays an important role in the regulation of smooth-muscle contraction. In this review, we will focus on the various Ca(2+)-transport processes that contribute to the cytosolic Ca2+ concentration. Mainly the functional aspects will be covered. The smooth-muscle inositol 1,4,5-trisphosphate receptor and ryanodine receptor will be extensively discussed. Smooth-muscle contraction also depends on extracellular Ca2+ and both voltage- and Ca(2+)-release-activated plasma-membrane Ca2+ channels will be reviewed. We will finally discuss some functional properties of the Ca2+ pumps that remove Ca2+ from the cytoplasm and of the Ca2+ regulation of the nucleus.

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice.

The fluorescent probe Fura-2/AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity

Connexin‐assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin‐hemichannel activity are unresolved. Here we investigated the regulation of Cx43‐hemichannel activity by actomyosin contractility and intracellular [Ca2+] ([Ca2+]i) using plasma membrane‐permeable TAT peptides (100 µM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy‐terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT‐Cx43CT) prevented the inhibition of Cx43‐hemichannel activity by contractility/high [Ca2+]i, whereas a reverse peptide (TAT‐Cx43CTrev) did not. These effects were independent of zonula occludens‐1, a cytoskeletal‐associated Cx43‐binding protein. In contrast, peptides corresponding to CL (TAT‐L2) inhibited Cx43‐hemichannel responses, whereas a mutant peptide (TAT‐L2H126K/I130N) did not inhibit. In these assays, TAT‐Cx43CT acted as a scaffold for TAT‐L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43‐hemichannel activity, because TAT‐Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C‐terminal tail (Cx43M239) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43‐hemichannel activity, laying the basis for developing hemichannel‐specific blockers.—Ponsaerts, R., De Vuyst, E., Retamal, M., Dhondt, C., Vermeire, D., Wang, N., De Smedt, H., Zimmermann, P., Himpens, B., Vereecke, J., Leybaert, L., Bultynck, G. Intramolecular loop/tail interactions are essential for connexin 43‐hemichannel activity. FASEB J. 24, 4378–4395 (2010). www.fasebj.org

Cytotoxicity and antiproliferative effect of hypericin and derivatives after photosensitization.

The toxicity on three human tumor cell lines (A431, HeLa and MCF7) of five phenanthroperylenequinones (hypericin and derivatives) and two perylenequinones (cercosporin and calphostin C) was investigated after photosensitization (4 J/cm2). Furthermore, the antiproliferative effect on HeLa cells was studied for the phenanthroperylenequinones. Hypericin, 2,5‐dibromohypericin, 2,5,9,12‐tetrabromohypericin and perylenequinones displayed a potent cytotoxic and antiproliferative effect in the nanomolar range. Hypericin dicarboxylic acid exhibited no photoactivity. In general, the antiproliferative activity correlated well with the photocytotoxicity. However, the nonphotocytotoxic compound hexamethylhypericin showed potent antiproliferative activity in the nanomolar range, probably exerting its action by protein kinase C inhibition. Without light irradiation, no cytotoxic and antiproliferative effect was observed for any photocytotoxic phenanthroperylenequinone compound. Furthermore, confocal laser microscopy revealed that the subcellular localization in A431 cells was similar for the photoactive compounds; the photosensitizers were mainly concentrated in the perinuclear region, probably corresponding with the Golgi apparatus and the endoplasmic reticulum. In addition, the accumulation of the photosensitizers in HeLa cells was investigated. All compounds except hypericin dicarboxylic acid were found to concentrate to a large extent in the cells. The compound 2,5,9,12‐tetrabromohypericin seemed intrinsically more effective than hypericin since the intracellular concentration of the bromoderivative was a magnitude of order lower than that of hypericin although both compounds showed similar photobiological activity.

Ca2+ Homeostasis in Vascular Smooth Muscle

The free intracellular calcium concentration is an important link in the excitation-contraction coupling mechanism of vascular smooth muscle. In this review, some current topics about vascular smooth muscle as regards Ca2+ storage, Ca2+ release, Ca2+ extrusion and Ca2+ regulation are discussed. Particular attention is paid to Ca2+ mobilized from the sarcoplasmic reticulum, the physiologically important Ca2+ reservoir in vascular smooth muscle. This occurs through two Ca2+ release channels: the inositol 1,4,5-trisphosphate receptor and the ryanodine receptor; the characteristics, function and control of these two receptors are summarized. Emphasis is also placed on a role of the nucleus as a potential Ca2+ storage site.

Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum.

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²(+), for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²(+)-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²(+) leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²(+) homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²(+) signalling.

Differential cytotoxic effects induced after photosensitization by hypericin

The cytotoxic effects of the natural photosensitizing agent hypericin were evaluated. A dramatic difference in the sensitivity of several different human and mouse cell lines towards photoactivated hypericin (4 J cm-2) was demonstrated using a neutral red assay (e.g. A431, IC50 = 0.14 +/- 0.02 microM; HeLa, IC50 = 0.32 +/- 0.05 microM, MCF7, IC50 = 1.84 +/- 0.22 microM). Dark cytotoxicity was absent, even at high hypericin concentration (25 microM). The differential phototoxicity of hypericin did not correlate with the expression of the epidermal growth factor (EGF) receptor nor with the expression of the P170 glycoprotein in the cell. The reduction of the intracellular glutathione content did not enhance further the cytotoxic effects of photoactivated hypericin, as investigated with the A431, HeLa and MCF7 cells. Conversely, using confocal laser microscopy, it was shown that the phototoxicity correlated well with the hypericin cellular uptake.