Repellency to ticks (Acari: Ixodidae) of extracts of Nigella sativa (Ranunculaceae) and the anti-inflammatory DogsBestFriend™.

Motivated by observations that the canine anti-inflammatory cream DogsBestFriend™ (DBF) appeared to deter flies, mosquitoes, and ticks from treated animals, repellent efficacy bioassays using four species of ticks were conducted with three extracts of Nigella sativa L. (Ranunculaceae), a constituent of DBF. The DBF cream was tested against nymphs of lone star tick, Amblyomma americanum (L.). In vertical filter paper assays, the three extracts applied at 0.413 mg extract/cm(2) filter paper repelled 96.7-100 % of brown dog tick, Rhipicephalus sanguineus (Latreille) nymphs, whereas, at the same rate, only one extract repelled >90 % A. americanum nymphs. Adult (mixed sexes) American dog ticks, Dermacentor variabilis (Say), required a higher concentration to be repelled effectively; two extracts, applied at 0.827 mg extract/cm(2) filter paper, repelled ≥90 % of the D. variabilis. In contrast, all extracts applied at much lower concentration (0.206 mg extract/cm(2) filter paper) repelled 100 % adult blacklegged ticks, Ixodes scapularis Say (only females tested). Of the two more repellent extracts, one lost most of its activity against A. americanum nymphs in <4 h when applied at 0.827 mg extract/cm(2) filter paper, whereas the other repelled 66.7 % of the nymphs at 192 h after application. At 0.206 mg extract/cm(2) filter paper, one extract was as repellent as deet against A. americanum nymphs. In a vertical bioassay in which nylon organdy was substituted for filter paper, DBF, at the rates of 1.67 and 0.835 mg cream/cm(2), repelled 76.7 and 30.0 % A. americanum nymphs, respectively. These findings indicate that when applied appropriately DBF should afford some protection to canines against tick bites.

Probing molecular identity of native single potassium channels by overexpression of dominant negative subunits.

Overexpression of dominant negative subunits previously has been shown to affect the whole cell delayed-rectifier potassium current (Ikv) in Xenopus spinal neurons. Here, we show that effects of overexpression of wild-type and dominant negative Kv1 channels are evident at the single channel level. The goal of these studies was to match molecular species of Kv subunits to specific, functionally identified single voltage-dependent potassium channels. In a heterologous system (the Xenopus oocyte), co-expression of wild-type and dominant negative mutant Kv1.1 subunits results in loss of active channels rather than channels of altered conductance. However, in situ overexpression studies are difficult to interpret due to the diversity in the control population of channels. Therefore, identification of endogenous channel populations containing Kv1 subunits is limited. Future work will reduce the endogenous diversity of potassium channels by study of single channels in identified subtypes of neurons.

Restoration of transient outward current by norepinephrine in cultured canine cardiac myocytes.

The mechanism for the reduction of the transient outward K+ current (Ito) in diseased myocardium is unknown. To identify potential mechanisms, the reduction of Ito and its subsequent restoration by norepinephrine (NE) were studied in cultured canine epicardial myocytes. After myocytes were cultured for 9 days (day 9), Ito density was decreased compared with density on the day of isolation (day 0) (3.2 +/- 0.4 vs. 10.4 +/- 0.4 pA/pF; mean +/- SE). The time constant of current decay (taudecay) was increased, the time course of recovery from inactivation was prolonged, and the half-inactivation voltage (V1/2) was shifted to less negative potentials. Exposure of myocytes on day 8 to 1 microM NE or isoproterenol (Iso) for 1 h had no acute effect on Ito but restored Ito density to 7.6 +/- 1.2 or 9.7 +/- 2.3 pA/pF, respectively, on day 9. Recovery from inactivation and taudecay remained slowed, and V1/2 remained shifted to less negative potentials. The effects of NE and Iso were blocked by actinomycin D and were not mimicked by phenylephrine or phorbol ester. A-23187 (1 microM) also restored Ito. Thus beta-adrenergic agonists restored normal Ito density, but not normal Ito kinetics, in cultured epicardial myocytes, possibly via increased intracellular Ca2+ concentration.

Developmental changes of action potential configuration and I(to) in canine epicardium.

Developmental changes of the transient outward current (I(to)) and action potential configuration were determined in canine epicardium ranging in age from fetal to 60 wk. The contributions of I(to) to rapid initial repolarization and to terminal repolarization were estimated by measuring the amplitude of phase 1 of the action potential and action potential duration, respectively. Phase 1 amplitude decreased progressively from fetal to 40 wk and remained constant thereafter. Action potential duration decreased from fetal to 2 wk, increased to 20 wk, and tended to decrease thereafter. Peak I(to) at +40 mV increased progressively from 2 to 60 wk. However, I(to) density was less at 2-10 wk than at 20-60 wk. The time constant of decay of I(to) increased with age from 2 to 60 wk, whereas the steady-state voltage dependence of inactivation did not vary with age. The time constant for the initial rapid phase of recovery from inactivation decreased from 2 to 10 wk and remained constant thereafter. The time constant for the more slowly evolving phase did not vary with age. The observation that the age-dependent reduction in phase 1 amplitude did not necessarily coincide with significant increases in I(to) density suggests that maturation of other ionic currents or transport mechanisms may contribute to developmental alterations of phase 1 repolarization.

Reduction of the transient outward potassium current in canine X-linked muscular dystrophy.

BACKGROUND: The xmd dog develops a cardiomyopathy similar to that seen in Duchenne muscular dystrophy patients. In both the canine and human diseases, ECG abnormalities may precede the development of overt cardiac pathological lesions. The purpose of this study was to determine whether specific cellular electrical abnormalities occur in dystrophic ventricular tissue. METHODS AND RESULTS: Action potentials were recorded in epicardial tissue strips obtained from normal and xmd dogs. Phase 1 amplitude was increased from 86.8 +/- 2.7 mV in normal dogs to 94.3 +/- 1.8 mV in xmd dogs (mean +/- SEM; P < .05). The 4-aminopyridine-sensitive transient outward potassium current (Ito), as recorded in isolated epicardial myocytes using the whole-cell patch-clamp technique, was reduced in xmd dogs compared with age-matched normal dogs. Cell capacitance also was reduced significantly in xmd compared with normal cells, as was the current density (3.6 +/- 0.3 versus 5.4 +/- 0.8 pA/pF, respectively). No differences were observed in the time constants of current decay or in the kinetics of recovery from inactivation between groups. The slope factor (k) of steady-state inactivation was significantly greater in xmd compared with normal cells (7.2 +/- 0.9 versus 5.4 +/- 0.5, respectively), whereas the V1/2 of inactivation did not differ (-38.2 +/- 2.4 versus -36.8 +/- 1.6 mV, respectively). CONCLUSIONS: These data indicate that the magnitude of Ito is reduced in dystrophic epicardial myocytes, resulting in an increase in phase 1 amplitude. The reduction of Ito may alter the balance of inward and outward currents in dystrophic myocardium and thereby contribute to the development of cardiac pathology.

Reduction of the transient outward potassium current in a canine model of Chagas' disease.

The effects of Chagas' disease, an important cause of cardiac arrhythmias and cardiomyopathy, on cellular electrical properties were determined in epicardial tissue from normal dogs and dogs infected with Trypanosoma cruzi for 20-25 days (25 DPI), at the time of maximum parasitemia, and for 125-140 days (140 DPI) after the parasitemia had subsided. At 25 DPI, phase 1 repolarization of the action potential was attenuated and the transient outward current (Ito) was reduced from 10.2 +/- 0.5 to 5.5 +/- 0.6 pA/pF. No differences were apparent between infected and normal cells in the time constants of current decay (25.6 +/- 4.0 and 22.8 +/- 1.3 ms, respectively) or in the steady-state inactivation parameters (V1/2 = -34.1 +/- 3.6 and -34.6 +/- 1.4 mV and k = 6.3 +/- 1.8 and 4.0 +/- 0.3, respectively). The rapid phase of recovery from inactivation was nearly eliminated in infected myocytes, whereas the slower phase was unaffected. Phase 1 repolarization and Ito density at 140 DPI were not significantly different from normal cells. Thus T. cruzi acutely inhibited Ito in epicardial myocytes, an effect that was reversed with abatement of the parasitemia.

Restoration of the transient outward potassium current by noradrenaline in chagasic canine epicardium.

1. The transient outward potassium current (Ito) is reduced in canine epicardial myocytes during the acute stage of infection with Trypanosoma cruzi (Chagas' disease). Sympathetic nerve terminals are also destroyed during the acute stage of this disease. To test whether the reduction of Ito is related to the absence of sympathetic innervation, acutely infected isolated epicardial myocytes were exposed in vitro to the sympathetic neurotransmitter noradrenaline (NA) and the effects of NA exposure on Ito were determined. 2. Continuous exposure to NA (1.0 microM) for 0-6 h had no effect on Ito density, whereas exposure to NA for 24 h significantly increased Ito density. Ito was also restored 24 h after a 1 h exposure to NA. Cell capacitance was not significantly affected by NA. 3. The alpha1-adrenergic receptor antagonist prazosin (0.1 microM) blocked the effects of NA on Ito, but the beta-adrenergic receptor antagonist propranolol (20 microM) did not. The beta-adrenergic receptor agonist isoprenaline (1 microM) had no effect on Ito. 4. Restoration of Ito by NA was prevented by pretreatment with neomycin (100 microM), a phospholipase C inhibitor, but not by pretreatment with 100-400 ng ml(-1) pertussis toxin (PTX). 5. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (0.1 microM) mimicked the effect of NA on Ito, whereas the inactive analogue 4alpha-phorbol (20 microM) had no effect on Ito. Pretreatment with bisindolylmaleimide (0.1 microM), a specific PKC inhibitor, completely blocked the effect of NA on Ito. 6. Thus, NA restores Ito in chagasic canine epicardial myocytes. The induction of Ito by NA appears to result from alpha1-adrenergic stimulation of PKC via a PTX-insensitive signalling cascade. These results suggest that the reduction of Ito in chagasic myocytes during the acute stage of Chagas' disease may reflect the lack of the trophic effects of sympathetic innervation.

Dietary taurine deficiency and dilated cardiomyopathy in the fox.

Taurine deficiency has been implicated as a potential cause of dilated cardiomyopathy. However, the relationship between taurine and myocardial function is presently unclear. The purpose of this study was to determine whether dilated cardiomyopathy in the fox is associated with dietary taurine deficiency. A total of 68 foxes from farms with a history of death caused by dilated cardiomyopathy and 14 foxes from a farm with no history of dilated cardiomyopathy were studied. Dilated cardiomyopathy was diagnosed by echocardiography in 48% of the foxes from one farm with a positive history and in none of the foxes from the control farm. Foxes less than 9 months of age were more commonly affected than older foxes (p = 0.03). Plasma taurine concentrations were significantly less (p less than 0.01) in foxes that had dilated cardiomyopathy (26.8 +/- 16.4 nmol/ml) than in the control foxes (99.3 +/- 60.2 nmol/ml). A significantly higher (p less than 0.01) incidence of dilated cardiomyopathy was present in foxes with a history of a sibling or offspring that died of dilated cardiomyopathy than in foxes without a family history of cardiac death. In one fox with dilated cardiomyopathy that was tested, the myocardial taurine concentration was lower (1.7 mumol/gm wet weight) than that of control foxes (7.3 +/- 1.6 mumol/gm wet weight). Hepatic cysteinesulfinic acid decarboxylase activity was significantly less (p less than 0.001) in foxes with dilated cardiomyopathy (0.97 +/- 0.2 nmol/mm.mg protein) than in control foxes (2.11 +/- 0.07 nmol CO2/mm.mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased density of Ito in left ventricular myocytes from German shepherd dogs with inherited arrhythmias.

INTRODUCTION: A colony of inbred German shepherd dogs with inherited ventricular arrhythmias has been established. METHODS AND RESULTS: The inward rectifier (IK1), the slow delayed rectifier (IKs), and the transient outward current (I(to)) were recorded from epicardial myocytes, and Ito was recorded from Purkinje myocytes isolated from the left ventricles of dogs mildly or severely affected with arrhythmias, and unaffected relatives. There were no differences between unaffected and severely affected dogs in the densities of either IK1 or IKs. Peak Ito density at +40 mV was reduced by 49% in epicardial myocytes from severely affected dogs. I(to) density was also reduced in a subset of Purkinje myocytes. Boltzmann analysis of steady-state inactivation showed no differences between groups in slope factor. V1/2, the half-inactivation voltage, was shifted by +6.2 mV in epicardial cells from severely affected versus unaffected dogs. In addition, the time constant for I(to) decay was reduced in mildly and severely affected dogs compared to unaffected dogs. CONCLUSION: Altered density and inactivation of I(to) are associated with the presence of severe ventricular arrhythmias in inbred dogs at risk for sudden death.