Jürgen F. Heubach

Electrophysiological properties of human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) have gained considerable interest due to their potential use for cell replacement therapy and tissue engineering. One strategy is to differentiate these bone marrow stem cells in vitro into cardiomyocytes prior to implantation. In this context ion channels can be important functional markers of cardiac differentiation. At present there is little information about the electrophysiological behaviour of the undifferentiated hMSC. We therefore investigated mRNA expression of 26 ion channel subunits using semiquantitative RT‐PCR and recorded transmembrane ion currents with the whole‐cell voltage clamp technique. Bone marrow hMSC were obtained from healthy donors. The cells revealed a distinct pattern of ion channel mRNA with high expression levels for some channel subunits (e.g. Kv4.2, Kv4.3, MaxiK, HCN2, and α1C of the L‐type calcium channel). Outward currents were recorded in almost all cells. The most abundant outward current rapidly activated at potentials positive to +20 mV. This current was identified as a large‐conductance voltage‐ and Ca2+‐activated K+ current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC50= 340 μm) and its inhibition by 100 nm iberiotoxin. A large fraction of cells also demonstrated a more slowly activating current at potentials positive to –30 mV. This current was selectively inhibited by clofilium (IC50= 0.8 μm). Ba2+ inward currents, stimulated by 1 μm BayK 8644 were found in a few cells, indicating the expression of functional L‐type Ca2+ channels. Other inward currents such as sodium currents or inward rectifier currents were absent. We conclude that undifferentiated hMSC express a distinct pattern of ion channel mRNA and functional ion channels that might contribute to physiological cell function.

Aconitum sp. alkaloids: The modulation of voltage-dependent Na+channels, toxicity and antinociceptive properties

Alkaloids from Aconitum sp., used as analgesics in traditional Chinese medicine, were investigated to elucidate their antinociceptive and toxic properties considering: (1) binding to Na+ channel epitope site 2, (2) alterations in synaptosomal Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), (3) arrhythmogenic action of isolated atria, (4) antinociceptive and (5) acute toxic action in mice. The study revealed a high affinity group (Ki 1 microM) and a low affinity group (Ki 10 microM) of alkaloids binding to site 2. The compounds of the high affinity group induce an increase in synaptosomal [Na+]i and [Ca2+]i (EC50 3 microM), are antinociceptive (ED50, 25 microg/kg), provoke tachyarrhythmia and are highly toxic (LD50 70 microg/kg), whereas low affinity alkaloids reduce [Ca2+]i, induce bradycardia and are less antinociceptive (ED50 20 mg/kg) and less toxic (LD50 30 mg/kg). These results suggest that the alkaloids can be grouped in Na+ channel activating and blocking compounds, but none of the alkaloids seem to be suitable as analgesics because of the low LD50/ED50 values.

Mode of antinociceptive and toxic action of alkaloids of Aconitum spec..

Abstract Extracts of the plant Aconitum spec. are used in traditional Chinese medicine predominantly as anti-inflammatory and analgesic agents, the latter allegedly equally potent as morphine but without any habit-forming potential. As the only pharmacologically active compounds, the C19 diterpenoid alkaloid aconitine, and some of its derivatives, have been proven to be antinociceptive in different analgesic assays, but the mode of action is unknown. To elucidate the mode of action, ten aconitine-like derivatives were investigated with respect to their affinity for voltage-dependent Na+ channels, the action on synaptosomal Na+ and Ca2+ homoeostasis and their antinociceptive, arrhythmogenic and acute toxic properties. Since aconitine is known to bind to site II of Na+ channels, we determined the affinity of the aconitine-like derivatives in vitro to synaptosomal membranes by the [3H]-batrachotoxinin-binding assay and their properties on intrasynaptosomal concentrations of free Na+ and Ca2+ ([Na+]i and [Ca2+]i), both the latter determined fluorometrically with SBFI and Fura-2 respectively. Furthermore, the alkaloids’ arrhythmogenic potential was investigated in guinea-pig isolated atria and the antinociceptive action on formalin-induced hyperalgesia and the acute toxic action estimated in mice. The results show that the alkaloids could be divided into at least three groups. The first is characterized by a high affinity to the site II of Na+ channels (Ki about 1.2 μM), the ability to enhance [Na+]i and [Ca2+]i (EC50 about 3 μM), a strong arrhythmogenic action that starts at about 30 nM, an antinociceptive effect (ED50 about 0.06 mg/kg) and high acute toxicity (LD50 values about 0.15 mg/kg). To this group belong aconitine, 3-acetylaconitine and hypaconitine. The second group, with lappaconitine as the only member, has an affinity to Na+ channels an order of magnitude lower (Ki = 11.5 μM), less acute toxicity (LD50 about 5 mg/kg), and a two orders of magnitude lower antinociceptive action (ED50 about 2.8 mg/kg) and lower cardiotoxicity (bradycardia observed at 3 μM). Additionally, lappaconitine suppresses the increase in [Ca2+]i of aconitine-stimulated synaptosomes and increases the excitation threshold of left atria, indicating an inhibition of Na+ channels. The other derivatives, i.e. delcorine, desoxydelcorine, karakoline, lappaconidine, lappaconine and lycoctonine, belong to the third group, which has hardly any effects. They have a low affinity to Na+ channels with Ki values in the millimolar range, show no effect on synaptosomal [Na+]i and [Ca2+]i, no arrhythmogenic potential up to 100 μM, no antinociceptive activity and low toxicity with LD50 values greater than 50 mg/kg. For the investigated alkaloids we suggest two different antinociceptive-like modes of action. Aconitine, hypaconitine and 3-acetylaconitine may induce a block of neuronal conduction by a permanent cell depolarisation, whereas lappaconitine might act like local anaesthetics. However, because of the low LD50/ED30 quotients of 2–6, the antinociceptive-like action of the Aconitum alkaloids seems to reflect severe intoxication rather than a specific antinociceptive action. The structure/activity relationship shows that alkaloids that activate or block Na+ channels have a benzoyl ester side chain in the C-14 or C-4 positions respectively, whereas the other compounds lack this group.

Molecular and Functional Expression of Voltage-Operated Calcium Channels During Osteogenic Differentiation of Human Mesenchymal Stem Cells

We used the patch‐clamp technique and RT‐PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L‐type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L‐type Ca2+ channel function.

Physiological antagonism between ventricular β1‐adrenoceptors and α1‐adrenoceptors but no evidence for β2‐ and β3‐adrenoceptor function in murine heart

Murine left atrium lacks inotropic β2‐adrenoceptor function. We investigated whether β2‐adrenoceptors are involved in the cardiostimulant effects of (−)‐adrenaline on spontaneously beating right atria and paced right ventricular myocardium of C57BL6 mice. We also studied a negative inotropic effect of (−)‐adrenaline. Sinoatrial tachycardia, evoked by (−)‐adrenaline was resistant to blockade by β2‐selective ICI 118,551 (50 nM) but antagonized by β1‐selective CGP 20712A (300 nM). This pattern was unaffected by pretreatment with pertussis toxin (PTX, 600 μg kg−1 i.p. 24 h) which reversed carbachol‐evoked bradycardia to tachycardia. Increases of ventricular force by (−)‐adrenaline and (−)‐noradrenaline were not blocked by ICI 118,551 but antagonized by CGP 20712A. Under blockade of β‐adrenoceptors, (−)‐adrenaline and (−)‐noradrenaline depressed ventricular force (−logIC50M=7.7 and 6.9). The cardiodepressant effects of (−)‐adrenaline were antagonized by phentolamine (1 μM) and prazosin (1 μM) but not by (−)‐bupranolol (1 μM). Prazosin potentiated the positive inotropic effects of (−)‐adrenaline (in the absence of β‐blockers) from −logEC50M=6.2–6.8. PTX‐treatment reduced carbachol‐evoked depression of ventricular force in the presence of high catecholamine concentrations. Inhibition of ventricular function of Gi protein was verified by 82% reduction of in vitro ADP‐ribosylation. PTX‐treatment tended to increase the positive inotropic potency of (−)‐adrenaline under all conditions investigated, including the presence of ICI 118,551. (−)‐Adrenaline causes murine cardiostimulation through β1‐adrenoceptors but not through β2‐adrenoceptors. The negative inotropic effects of (−)‐adrenaline are mediated through ventricular α1‐adrenoceptors but not through β3‐adrenoceptors. Both Gi protein and α1‐adrenoceptors restrain (−)‐adrenaline‐evoked increases in right ventricular force mediated through β1‐adrenoceptors.

Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an “artificial niche” of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin‐positive cells (intestinal epithelium‐derived nestin‐positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP‐responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic‐like stage of development that has a high degree of flexibility and plasticity.

Effects of azelastine on contractility, action potentials and L-type Ca2+ current in guinea pig cardiac preparations

Azelastine is used for symptomatic relief of allergic rhinitis and asthma bronchiale. In vitro studies in smooth muscle cells from guinea pig trachea and ileum demonstrate that the drug blocks L-type Ca(2+) current (I(Ca, L)). However, for safety reasons, it is important to know whether azelastine also affects cardiac I(Ca, L) in therapeutically relevant concentrations. We have therefore studied the effects of azelastine on I(Ca, L) in guinea pig ventricular myocytes using standard whole-cell patch-clamp technique. Force of contraction and action potentials from isolated papillary muscles of the same species were also investigated at physiological temperature (36 degrees C). Azelastine (30 microM) significantly reduced force of contraction, shortened action potential duration, and depressed maximum upstroke velocity. I(Ca, L) was elicited by 200-ms-long clamp steps from -100 to 0 mV (one pulse every 3 s). Azelastine blocked I(Ca, L) reversibly and concentration-dependently with an IC(50) of 20.2+/-1.3 microM and a Hill coefficient of 1.1. At 10 microM, azelastine shifted steady-state inactivation by 5 mV (n=7) to more negative potentials. The time course of I(Ca, L) inactivation could be described by a double exponential function. Azelastine (10 microM) significantly shortened the slow inactivation time constant (tau(s)) from 54.2+/-2.8 ms under control conditions to 38.7+/-2.9 ms (n=16) in the presence of drug. Azelastine also reduced low-voltage-activated Ca(2+) currents with a similar IC(50) value (24 microM, at -35 mV). Since the therapeutic plasma concentrations are in the order of 10-100 nM, we conclude that azelastine does indeed affect also cardiac I(Ca, L), but the concentrations required are at least two orders of magnitude larger than those obtained during drug therapy.