Lupin (Lupinus spp.) seeds exert anthelmintic activity associated with their alkaloid content.

The growing range of drug resistant parasitic nematode populations threatens the sustainability of ruminant farming worldwide. In this context, nutraceuticals, animal feed that provides necessary dietary requirements while ensuring parasite control, could contribute to increase farming sustainability in developed and low resource settings. In this study, we evaluated the anthelmintic potential of lupin seed extracts against the major ruminant trichostrongylids, Haemonchus contortus and Teladorsagia circumcincta. In vitro observations showed that seed extracts from commercially available lupin varieties could significantly but moderately inhibit larval migration. This anthelmintic effect was mediated by the seed alkaloid content and was potent against both fully susceptible and multidrug resistant H. contortus isolates as well as a susceptible T. circumcincta isolate. Analytical chemistry revealed a set of four lupanine and sparteine-derivatives with anthelmintic activity, and electrophysiology assays on recombinant nematode acetylcholine receptors suggested an antagonistic mode of action for lupin alkaloids. An in vivo trial in H. contortus infected lupin-fed ewes and goats failed to demonstrate any direct anthelmintic effect of crude lupin seeds but infected lupin-fed goats suffered significantly less parasite-mediated blood losses. Altogether, our findings suggest that the anthelmintic potential of lupin remains limited. However, the potent alkaloids identified could lead to the development of novel drugs or may be used in combination with current anthelmintics to improve their efficacy.

cGMP-mediated inhibition of cardiac L-type Ca(2+) current by a monoclonal antibody against the M(2) ACh receptor.

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M(2) receptor were studied on the L-type Ca(2+) currents (I(Ca,L)) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I(Ca,L) but did not significantly affect basal I(Ca,L). Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I(Ca,L) were not modified in presence of B8E5. Inhibition of I(Ca,L) by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase [1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one]. These results indicate that the antibody B8E5 inhibits the beta-adrenergic-stimulated I(Ca,L) through activation of the M(2) muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.

Calcium-dependent modulation of the plateau phase of action potential in isolated ventricular cells of rabbit heart.

[Ca2+]i-dependent modulation of the action potential has been studied in Fura-2 dialysed ventricular myocytes of the rabbit using the whole-cell current-clamp method. Fifteen consecutive action potentials (AP1-AP15) and [Ca2+]i transients were elicited at a frequency of 0.2 Hz. A single, brief application of caffeine (during AP9) first enhanced and thereafter attenuated the [Ca2+]i transients accompanying AP9 and AP10-AP12, respectively. This approach provided direct comparison between time courses of action potentials: during the initial steady state (e.g. AP8) and when Ca2+ release from the sarcoplasmic reticulum was increased by caffeine (AP9) or decreased by depletion (AP10). The increase in [Ca2+]i facilitated repolarization and decreased action potential duration. However, action potentials at reduced Ca2+ release (AP10) had longer duration than during steady state. The caffeine-induced changes in L-type Ca2+ current (ICa,L), during voltage-clamp conditions partially explained the effects of caffeine on action potentials. When ICa,L was blocked by 500 micromol L-1 Cd2+, enhanced [Ca2+]i transients revealed an extra current component which was outward at +10 mV and inward at the resting membrane potential (most probably the transient inward current). In the presence of Cd2+, however, AP8 and AP10 had identical time courses, suggesting that ICa,L alone was responsible for the lengthening of AP10. Alterations in the transmembrane Na+ gradient resulted in changes of the steady state action potential durations (AP8) consistently with the expected modulation of the Na+-Ca2+ exchange current. However, the contribution of this current to the [Ca2+]i-dependent behaviour of action potential plateau could not be demonstrated.

In vivo and in vitro inhibition of the L-type calcium current in isolated guinea-pig cardiomyocytes by the immunosuppressive agent cyclosporin A.

Cyclosporin A (CsA), an immunosuppressive agent used to reduce rejection after organ transplantation, induces secondary effects in heart tissue. We have studied the effects in vivo and in vitro of CsA on L-type Ca2+ current (ICa) and the associated gating currents of isolated guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. For in vivo experiments, a group of animals (n=28) was treated for 21 days by subcutaneous injection of CsA (15 mg/kg/day). Blood level of CsA was 1191+/-221 ng/ml (n=9). In cells from these animals (n=65, 19 animals), ICa was reduced to about 75% of that recorded from control cells (n=32, six animals). CsA decreased the availability of Ca2+ channels at potentials more positive than +30 mV. Isoproterenol (100 nM) was still able to increase ICa but only by 30+/-6% (n=9), whereas in control it increased ICa by 290+/-22% (n=5). Gating currents related to L-type Ca2+ channels were not altered in cells from CsA-treated animals. In in vitro experiments, CsA reduced ICa when applied directly to cardiomyocytes. CsA affected the kinetics of ICa inactivation, slowing down the rapid phase and accelerating the slow phase (n=4). The steady-state inactivation curve of ICa was shifted to more negative voltages and the degree of availability at -80 mV decreased by in vitro application of CsA. The half inactivation potential (V1/2) changed from -23+/-0.6 mV in control to -31+/-2 mV, -48+/-0.6 mV and -49+/-0.6 mV, in 1, 50 and 80 microM CsA, respectively. In these cells, the gating currents related to L-type Ca2+ channels were also not altered by CsA. CsA does not modify the Ca2+ channel density, although it induces a decrease in the beta1-adrenergic stimulation of ICa. The results are explained by a direct effect on the calcium channel inactivation of CsA and a non specific indirect effect.

Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the beta 1-adrenergic and M2-muscarinic receptors and regulate calcium channels in isolated cardiomyocytes.

Sera from T. cruzi infected mice were tested in an enzyme immunoassay on peptides corresponding to the second extracellular loops of the beta 1-, the beta 2-adrenergic receptor and the M2 muscarinic receptor. All sera of mice (4/4) in the acute phase recognized the beta 1-adrenergic receptor and the M2 muscarinic receptor peptides but not the beta 2-adrenergic receptor peptide. The same peptides were recognized during the chronic phase in half of the mice (6/12). The immunoglobulin fractions of the mice were tested for their activity on L-type Ca++ channels of isolated guinea-pig cardiomyocytes using the whole-cell patch clamp technique. The immunoglobulin fractions of acute phase mice were able to activate the Ca++ channels by stimulation of the beta-adrenergic receptors, as assessed by inhibition with propranolol. Those of the chronic phase mice reduced the Ca++ current by stimulation of the muscarinic receptors, as assessed by inhibition with atropine. These results confirm the existence of functional epitopes on the second extracellular loops of both receptors. They suggest that, as in humans, the parasite is able to elicit functional autoantibodies against these epitopes. They give evidence that these autoantibodies mediate their physiological effects by modulating the cAMP activated Ca++ channels.

Effect of sulfhydryl oxidation on ionic and gating currents associated with L-type calcium channels in isolated guinea-pig ventricular myocytes.

OBJECTIVE: L-type calcium currents (ICa) and gating currents modification by extracellular application of the selective free sulfhydryl oxidant p-hydroxy-mercuric-phenylsulphonic acid (PHMPS) were studied. METHODS: Both currents were obtained with the whole cell patch clamp technique in guinea-pig ventricular cardiocytes. RESULTS: The main finding was a reduction of ICa clearly differentiable from a "run down" process. This effect was protected, stopped and in some cases partially reversed by dithiothreitol, a protective reagent for -SH groups. We also found a decrease of the gating currents associated with L-type calcium channels. The calcium modulation and cAMP phosphorylation systems of ICa are unaffected by PHMPS. With barium as charge carrier the current-voltage curves of barium currents were shifted by 10 mV to the positive direction by PHMPS. The same effect was obtained with calcium currents using BAPTA as a fast calcium buffer. CONCLUSION: The results indicate that oxidation of -SH groups carried by the channel protein induces dysfunction of the calcium entry to cardiac cells by altering the gating process. A participation of thiol functions on the gating of the calcium channel is proposed.

A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation.

A mixture of crude collagenase and non-specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünsch's substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS-PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium-tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.

Alteration of the L-type calcium current in guinea-pig single ventricular myocytes by heptaminol hydrochloride.

1. The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea-pig by use of the whole cell configuration of the patch clamp technique. 2. A concentration-dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10(-6) M, this effect was observed in only two cells (n = 6). At 10(-5) M the reduction of ICa was of 30 +/- 15% (n = 11). 3. The current recovery from inactivation at -40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (greater than 10(-6) M). However, at -80 mV HP the overshoot of the recovery curve was decreased by heptaminol. 4. Both at -40 mV and -80 mV HP, heptaminol (10(-5) M) significantly increased the steady state inactivation of ICa. 5. As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.

Rate dependence of action potential duration and calcium current in isolated guinea-pig cardiocytes.

The duration of the action potential at 50% of its amplitude (APD50) and peak calcium currents (ICa) was measured in single cardiac guinea-pig ventricular cells, using the whole-cell patch-clamp technique in current-clamp and voltage-clamp modes respectively. In the absence of intracellular calcium buffer, pacing at 0.28 Hz from a rest period of 1-2 min induced a transient increase (15.5 +/- 3.5%) in APD50, followed by a gradual shortening. Switching from 0.28 to 0.75 Hz again induced a transient increase of APD50 (6.8 +/- 2.9%). In the presence of EGTA or BAPTA on the cytosolic side of the membrane, this transient phase was prolonged and its amplitude slightly increased (10.6% when switching from 0.28 to 0.75 Hz in 5 mM-BAPA). The same increase in rate induced either a negative or a positive staircase of ICa, depending on the holding potential. At a holding potential of -80 mV, ICa peak was enhanced and the inactivation kinetics was slowed down. This facilitation of ICa seems to be dependent on calcium ions entering the cell via the calcium channels and could partly explain the observed transient increase in APD50.

A new method of attachment of isolated mammalian ventricular myocytes for tension recording: length dependence of passive and active tension.

The study of the Frank-Starling's law in mammalian single cells has been hindered by a lack of an easily performed method of stretching cells. Some authors have succeeded in this but their methods required a great deal of technical expertise and in most cases they have not had much success. We have developed an easy method of stretching mammalian ventricular cells from slack sarcomere length (S.L.) (Lo, 1.77 +/- 0.05 microns) to about 117% of this length. Thin carbon fibers (12 microns in diameter) which can be bound electrochemically to the cell membrane surface have been used. A flexible long fiber of known compliance (80 microns/microN) was attached to one end of the cell and a stiff double fiber (4 microns/microN) to the other end. The cell attachment was relatively easy to perform and successful results were obtained in 80% of the attempts. The displacement of the flexible fiber allows the quantitative measurements of the resting tension in a group of non-stimulated cells and of auxotonic contractions developed upon stimulation in another group of cells. Increasing S.L. from Lo to 105-106% of Lo, an increase in active tension from 0.21 +/- 0.03 mN/mm to 0.26 +/- 0.01 mN/mm (n = 4) could be noticed with a stimulation frequency of 0.5 Hz. An increase in active tension was also observed at 1 Hz. Staircase kinetics were accelerated with stretching; this confirms at the single cell level the hypothesis of an effect of length-dependent activation on the staircase. Eulerian differential stiffness constant was calculated and found to be 13.5 +/- 1.2, a value which is comparable to that described in intact heart. Thus the important stiffness found in the whole heart may be due to intracellular component(s) such as myofilament and/or connectin.