Tissue engineering of a differentiated cardiac muscle construct.

Cardiac tissue engineering is an emerging field. The suitability of engineered heart tissue (EHT) for both in vitro and in vivo applications will depend on the degree of syncytoid tissue formation and cardiac myocyte differentiation in vitro, contractile function, and electrophysiological properties. Here, we demonstrate that cardiac myocytes from neonatal rats, when mixed with collagen I and matrix factors, cast in circular molds, and subjected to phasic mechanical stretch, reconstitute ring-shaped EHTs that display important hallmarks of differentiated myocardium. Comparative histological analysis of EHTs with native heart tissue from newborn, 6-day-old, and adult rats revealed that cardiac cells in EHTs reconstitute intensively interconnected, longitudinally oriented, cardiac muscle bundles with morphological features resembling adult rather than immature native tissue. Confocal and electron microscopy demonstrated characteristic features of native differentiated myocardium; some of these features are absent in myocytes from newborn rats: (1) highly organized sarcomeres in registry; (2) adherens junctions, gap junctions, and desmosomes; (3) a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum; and (4) a basement membrane surrounding cardiac myocytes. Accordingly, EHTs displayed contractile characteristics of native myocardium with a high ratio of twitch (0.4 to 0.8 mN) to resting tension (0.1 to 0.3 mN) and a strong beta-adrenergic inotropic response. Action potential recordings demonstrated stable resting membrane potentials of -66 to -78 mV, fast upstroke kinetics, and a prominent plateau phase. The data indicate that EHTs represent highly differentiated cardiac tissue constructs, making EHTs a promising material for in vitro studies of cardiac function and tissue replacement therapy.

The hyperpolarization-activated current If in ventricular myocytes of non-transgenic and beta2-adrenoceptor overexpressing mice.

In transgenic mice (TG4) overexpressing the human beta2-adrenoceptor (beta2-AR), unoccupied receptors are supposed to activate spontaneously the signalling cascade, leading to enhanced levels of cAMP. This second messenger shifts activation curves of the hyperpolarization-activated current If towards less negative potentials. Here, we characterize If of ventricular myocytes from non-transgenic littermate (LM) and TG4 mice and investigate whether If is modulated by spontaneous beta2-AR signalling. If was activated in whole-cell voltage-clamp experiments during test steps ranging from -65 mV to -135 mV (holding potential: -55 mV; 36 degrees C). In TG4 the maximum amplitude was fivefold larger than in LM myocytes (-1.10 +/- 0.11 pA/pF vs. -0.22 +/- 0.04 pA/pF at -135 mV), and the potential for half-maximum If current (VI0.5) was less negative (-100.5 +/- 1.0 mV in TG4 vs. -108.4 +/- 2.6 mV in LM). (-)-Isoproterenol (1 microM) shifted VI0.5 of LM myocytes by 10.4 mV towards less negative potentials but had no significant effect in TG4. However, the inverse beta2-AR agonist ICI 118,551 (300 nM) shifted VI0.5 of TG4 myocytes to values observed in LM under control conditions, suggesting a relation to spontaneously active beta2-ARs. Enhanced expression of hyperpolarization-activated and cyclic nucleotide gated channels (HCN) could contribute to increased maximum If amplitude in TG4 myocytes. Semi-quantitative RT-PCR analysis demonstrated a 1.8-fold elevation of HCN4 mRNA and no significant change for HCN2 mRNA in TG4 ventricle. Cardiac hypertrophy was not detected in TG4 mice investigated here. We conclude that spontaneous beta2-AR signalling in hearts of TG4 mice shifts If current-voltage relation towards less negative potentials. Increased maximum If amplitude in TG4 myocytes is in line with enhanced expression of HCN channels. Both mechanisms could contribute to larger inward current at physiological diastolic potentials.

Effects of azelastine on contractility, action potentials and L-type Ca(2+) 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.

Murine ventricular L-type Ca(2+) current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor.

1. The functional coupling of beta(2)-adrenoceptors (beta(2)-ARs) to murine L-type Ca(2+) current (I(Ca(L))) was investigated with two different approaches. The beta(2)-AR signalling cascade was activated either with the beta(2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta(2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta(2)-ARs). Ca(2+) and Ba(2+) currents were recorded in the whole-cell and cell-attached configuration of the patch-clamp technique, respectively. 2. Zinterol (10 microM) significantly increased I(Ca(L)) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta(1)-AR subtype, since it was blocked by the beta(1)-AR selective antagonist CGP 20712A (300 nM). The beta(2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I(Ca(L)) to zinterol. 3. In myocytes with beta(2)-AR overexpression I(Ca(L)) was not stimulated by the activated signalling cascade. On the contrary, I(Ca(L)) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I(Ca(L)). The beta(2)-AR inverse agonist ICI 118,551 did not further decrease I(Ca(L)). PTX-treatment increased current amplitude to values found in control myocytes. 4. In conclusion, there is no evidence for beta(2)-AR mediated increases of I(Ca(L)) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta(2)-AR responses to zinterol, but augments beta(1)-AR mediated increases of I(Ca(L)). In the mouse model of beta(2)-AR overexpression I(Ca(L)) is reduced due to tonic activation of Gi-proteins.

Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima.

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

The effect of Gi-protein inactivation on basal, and beta(1)- and beta(2)AR-stimulated contraction of myocytes from transgenic mice overexpressing the beta(2)-adrenoceptor.

The atria and ventricles of transgenic mice (TGbeta(2)) with cardiac overexpression of the human beta(2)-adrenoceptor (beta(2)AR) were initially reported to show maximum contractility in the absence of beta-AR stimulation. However, we have previously observed a different phenotype in these mice, with myocytes showing normal contractility but reduced betaAR responses. We have investigated the roles of cyclic AMP and Gi in basal and betaAR function in these myocytes. ICI 118,551 at inverse agonist concentrations decreased contraction by 32%. However, the cyclic AMP antagonist Rp-cAMPS had no effect on contraction in TGbeta(2) myocytes, indicating that there was no tonic influence of raised cyclic AMP. These findings cannot be explained by the proposed model for inverse agonism, where the activated receptor (R*) raises cyclic AMP levels and so increases contraction in the absence of agonist. After pertussis toxin (PTX) pretreatment to produce inactivation of Gi, the basal contraction in 1 mM Ca(2+) was increased in TGbeta(2) mice (7.82+/-0.47%, n=23) compared to LM mice (3.60+/-0.59%, n=11) (P<0.001). The contraction amplitude of myocytes to the maximal concentration of isoprenaline was also increased significantly by PTX in TGbeta(2) mice (9.40+/-1.22%, n=8) and was no longer reduced compared to LM mice (8.93+/-1.50%, n=11). Both beta(1)- and beta(2)AR subtypes were affected both by the original desensitization and by the resensitization with PTX. PTX treatment has therefore restored the original phenotype, with high basal contractility and little further effect of isoprenaline. We suggest that both beta-AR desensitization and lack of increased basal contraction in ventricular myocytes from our colony of TGbeta(2) mice were due to increased activity of PTX-sensitive G-proteins.

T-Type and tetrodotoxin-sensitive Ca(2+) currents coexist in guinea pig ventricular myocytes and are both blocked by mibefradil.

Under Na(+)-free conditions, low-voltage-activated Ca(2+) currents in cardiomyocytes from various species have been described either as Ni(2+)-sensitive T-type Ca(2+) current (I(Ca(T))) or as tetrodotoxin (TTX)-sensitive Ca(2+) current (I(Ca(TTX))). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which I(Ca(T)) and I(Ca(TTX)) can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na(+)-free solutions. Whereas rat myocytes lack I(Ca(T)) and exhibit I(Ca(TTX)) only, guinea pig myocytes possess both of these low-voltage-activated Ca(2+) currents, which are separated pharmacologically by superfusion with TTX or Ni(2+). I(Ca(T)) and I(Ca(TTX)) were of similar amplitude but significantly differed in their electrophysiological properties: I(Ca(TTX)) activated at more negative potentials than did I(Ca(T)), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. I(Ca(TTX)) but not I(Ca(T)) increased after membrane rupture ("run-up"). Isolation of I(Ca(TTX)) by application of the bivalent cation Ni(2+) is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca(2+) channel blocker mibefradil (10 micromol/L) is a suitable tool for the study of I(Ca(TTX)). However, mibefradil not only blocked I(Ca(T)) by 85+/-2% but also decreased I(Ca(TTX)) by 48+/-8%. We conclude that under Na(+)-free conditions I(Ca(T)) and I(Ca(TTX)) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of I(Ca(T)) will have to consider the TTX-sensitive current component to avoid possible interference.

Beta4-adrenoceptors are more effective than beta1-adrenoceptors in mediating arrhythmic Ca2+ transients in mouse ventricular myocytes.

Putative beta4-adrenoceptors mediate cardiostimulation and arrhythmias in mammalian heart. Both beta1- and putative beta4-adrenoceptors mediate arrhythmias but through different mechanisms. To elucidate further the mechanisms of cardiostimulation and arrhythmias we measured Ca2+ transients and L-type Ca2+ currents in mouse ventricular myocytes. We used (-)-CGP 12177, an antagonist of beta1- and beta2-adrenoceptors with agonist properties at the putative beta4-adrenoceptor, and (-)-isoprenaline as an agonist for beta1- and beta2-adrenoceptors. (-)-CGP 12177 increased Ca2+ transients in electrically stimulated cells loaded with Indo-1. The maximum increase of Ca2+ transients caused by (-)-CGP 12177 amounted to approximately one-third of that caused by maximally effective (-)-isoprenaline concentrations. Both (-)-CGP 12177 and (-)-isoprenaline caused concentration-dependent arrhythmic Ca2+ transients. The arrhythmias appeared at paced Ca2+ transients and between paced Ca2+ transients. The arrhythmic potency of (-)-CGP 12177 (-logEC50=9.4) was approximately 40 times greater than that of (-)-isoprenaline (-logEC50=7.8). L-type Ca2+ current was measured in the whole cell configuration of the patch clamp technique. In the presence of both 3-isobutyl 1-methylxanthine (6 micromol/l) and (-)-propranolol (500 nmol/l), (-)-CGP 12177 (100 nmol/l) increased significantly L-type Ca2+ current by 19% of the effect of (-)-isoprenaline. The (-)-CGP 12177-evoked increase of Ca2+ transients contrasts with the smaller effects on L-type Ca2+ current, suggesting that activation of the putative beta4-adrenoceptor causes a more efficient Ca2+-induced Ca2+ release than activation of the beta1-adrenoceptor. Beta4-Adrenoceptors mediate arrhythmias with smaller Ca2+ transients and smaller increases of L-type Ca2+ current than beta1-adrenoceptors, in line with different but still unknown mechanisms as previously suggested for the intact heart.

L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac beta 2-adrenoceptor.

OBJECTIVES: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human beta 2-adrenoceptor (beta 2-AR) was explained by spontaneously active beta 2-ARs that stimulate the beta-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls. METHODS: Cardiac beta-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria. RESULTS: Overexpression of beta 2-ARs was confirmed by an almost 400-fold increase in beta-AR density. The beta 1:beta 2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o -4.82 +/- 0.48 vs. -6.56 +/- 0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 microM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice. CONCLUSIONS: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating beta 2-ARs in left atrium and ventricle. A contractile deficit unrelated to the beta-adrenoceptor pathway is evident in TG4 myocytes and left atria.

Cardiac effects of lappaconitine and N-deacetyllappaconitine, two diterpenoid alkaloids from plants of the Aconitum and Delphinium species.

Aconitum and Delphinium alkaloids are currently under investigation in search for new analgesic and anti-inflammatory drugs. It has been reported that the analgesic compound lappaconitine (LA), a C19 diterpenoid alkaloid from Aconitum sinomontanum nakai is an inhibitor of tetrodotoxin-sensitive, voltage-dependent sodium channels. In the present study we investigated the cardiac effects of LA and its metabolite N-deacetyllappaconitine (DLA) in electrically stimulated left and spontaneously beating right atria isolated from guinea-pig hearts. In all experiments, equieffective concentrations were larger with DLA than with LA. At a stimulation frequency of 2.5 Hz the time constant for the onset of LA effects (tau = 56 +/- 29 min) was markedly larger than the one for DLA effects (tau = 14 +/- 8 min). The compounds exerted a significant negative inotropic action at 0.06 microM (LA) and 0.2 microM (DLA). Asystolia of right atria occurred at 4.5 microM (LA) and 10 microM (DLA). Therefore, cardiotoxicity of LA and DLA was much lower compared to aconitine, which caused arrhythmia at 10 nM in our model. For both alkaloids a use-dependent mode of action could be demonstrated. In addition, preincubation with 0.3 microM LA prevented arrhythmia induced by aconitine or ouabain. We conclude that lappaconitine is a naturally occurring compound with class-I antiarrhythmic action.

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

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 microM), the ability to enhance [Na+]i and [Ca2+]i (EC50 about 3 microM), 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 microM), 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 microM). 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 microM, 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.

Inhibition of cross-bridge binding to actin by caldesmon fragments in skinned skeletal muscle fibers.

Several regions within the 35-kDa COOH-terminal portion of caldesmon have been implicated in the ability of caldesmon to inhibit actin-activated myosin ATPase activity. To further define the functional regions of caldesmon, we have studied the effects of three chymotryptic fragments, one fragment produced by CNBr digestion and two fragments produced by digestion with submaxillaris arginase C protease, on the relaxed stiffness and active force of rabbit psoas fibers. Each of the regions of caldesmon studied had either direct or indirect effects on single-fiber mechanics. The 35-kDa and 20-kDa fragments of caldesmon, like intact caldesmon, were effective inhibitors of fiber stiffness, a measure of cross-bridge attachment. The 7.3-kDa and 10-kDa fragments, which constitute the NH2 and COOH halves of the 20-kDa fragment, inhibited both relaxed fiber stiffness and active force production, but with a reduced efficacy compared to the 20-kDa fragment. These results suggest that several regions within the 35-kDa COOH-terminal region of caldesmon are required for optimum function of caldesmon and that function includes inhibition of weak cross-bridge attachment and force production.

6-Benzoylheteratisine - a class-I antiarrhythmic substance from Aconitum tanguticum (Maxim.) Stapf.

The antiarrhythmic action of 6-benzoylheteratisine (6-bh), a C(19) diterpenoid alkaloid from Aconitum tanguticum (Maxim.) Stapf was investigated in left and right guinea pig isolated atria. Furthermore, possible effects on transmembrane action potential of isolated papillary muscles were studied using microelectrode techniques. At concentrations of more than 6 × 10(-8) mol/1, preincubation with 6-bh suppressed arrhythmias induced by aconitine, veratridine and ouabain. Bradycardia of the right atria as a sign of toxicity occurred at 1 × 10(-6) mol/1. The alkaloid significantly reduced the maximum rate of rise of the action potential as well as the action potential amplitude, indicating inhibition of voltage-dependent sodium channels as a functional principle. Additionally, a use-dependent mode of drug-action could be demonstrated. We conclude therefore, that 6-bh is a naturally occurring class-I antiarrhythmic substance. The compound is a main alkaloid of Aconitum tanguticum, a plant used to prepare a poison antidote in Chinese and Tibetan folk medicine. It may be speculated that the poison antidote effect is at least partially based on the antiarrhythmic properties of 6-bh.

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.