Jader Santos Cruz

Impairment of In Vitro and In Vivo Heart Function in Angiotensin-(1-7) Receptor Mas Knockout Mice

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas−/− mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4±0.09 versus 2.1±0.03 g in Mas+/+ mice), ±dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart.

Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/KATP signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kγ/AKT protein kinase B (AKT) and culminated in increased activation of KATP channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents

Citronellol is one monoterpene alcohol, which is present in the essential oils of various aromatic plant species. This study evaluated the neuroprotective activity of citronellol on pentylenetetrazol- and picrotoxin-induced convulsions and maximal electroshock-induced seizures in mice. Administration of citronellol significantly reduced the number of animals of convulsion induced by pentylenetetrazol and eliminated the extensor reflex of maximal electroshock-induced seizures test in about 80% of the experimental animals. In addition, administration of citronellol showed protection in the pentylenetetrazol and picrotoxin tests by increasing the latency of clonic seizures. We also investigated the effect of citronellol in the rat isolated nerve using the single sucrose-gap technique. We showed that the amplitude of the compound action potential decreased more than 90% when the monoterpene was incubated for 30 min at 6.4 mM and we did not verify any effect on the repolarization of the compound action potential. Taken together, our results demonstrated an anticonvulsant activity of the citronellol that could be, at least in part, explained by the diminution of the action potential amplitude.

Functional and structural features of γ-zeathionins, a new class of sodium channel blockers

γ1‐ and γ2‐zeathionins (γ1‐Z and γ2‐Z) are members of a family of small and basic peptides involved in plant protection. These plant defensins exhibit remarkable structural similarity to scorpion neurotoxins and insect defensins. In the present report, we used the whole‐cell patch clamp technique to investigate the inhibition of the sodium current (I Na) by γ1‐Z and γ2‐Z in the GH3 cell line. Both γ1‐Z and γ2‐Z rapidly and reversibly inhibited I Na without changing the kinetics or voltage dependence of activation or inactivation. To our knowledge, this is the first example of a plant protein that inhibits the sodium channel. From structural comparisons with the μ‐conotoxins, a family of peptides that block the sodium channel, we detected some similar features that could provide the basis of inhibition of sodium channels by γ‐zeathionins.

Phoneutria nigriventer toxin Tx3-1 blocks A-type K+ currents controlling Ca2+ oscillation frequency in GH3 cells.

Abstract: GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3‐1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3‐1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4‐aminopyridine and charybdotoxin. Whole‐cell patch clamp experiments show that Tx3‐1 reversibly inhibits the A‐type K+ current (IA) but does not block other K+ currents (delayed‐rectifying, inward‐rectifying, and large‐conductance Ca2+‐sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3‐1, which shows that Tx3‐1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3‐1 is a selective inhibitor of IA, which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3‐1, we suggest that IA is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1 The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3‐3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2 Tx3‐3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3‐3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1‐43. 3 Using ω‐toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3‐3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage‐sensitive calcium channels altered by Tx3‐3 can also be inhibited by ω‐agatoxin IVA, an antagonist of P/Q calcium channels. ω‐conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4 It is concluded that Tx3‐3 potently inhibits ω‐agatoxin IVA‐sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Dysautonomia Due to Reduced Cholinergic Neurotransmission Causes Cardiac Remodeling and Heart Failure

ABSTRACT Overwhelming evidence supports the importance of the sympathetic nervous system in heart failure. In contrast, much less is known about the role of failing cholinergic neurotransmission in cardiac disease. By using a unique genetically modified mouse line with reduced expression of the vesicular acetylcholine transporter (VAChT) and consequently decreased release of acetylcholine, we investigated the consequences of altered cholinergic tone for cardiac function. M-mode echocardiography, hemodynamic experiments, analysis of isolated perfused hearts, and measurements of cardiomyocyte contraction indicated that VAChT mutant mice have decreased left ventricle function associated with altered calcium handling. Gene expression was analyzed by quantitative reverse transcriptase PCR and Western blotting, and the results indicated that VAChT mutant mice have profound cardiac remodeling and reactivation of the fetal gene program. This phenotype was attributable to reduced cholinergic tone, since administration of the cholinesterase inhibitor pyridostigmine for 2 weeks reversed the cardiac phenotype in mutant mice. Our findings provide direct evidence that decreased cholinergic neurotransmission and underlying autonomic imbalance cause plastic alterations that contribute to heart dysfunction.

Inhibition of neuronal high-voltage activated calcium channels by the ω-Phoneutria nigriventer Tx3-3 peptide toxin

We have investigated the effect of omega-PnTx3-3 (referred to in previous papers simply as Tx3-3), a peptide toxin from the venom of the spider Phoneutria nigriventer, on neuronal high-voltage activated (HVA) Ca(2+) channels, using whole-cell patch-clamp. omega-PnTx3-3 (120 nM) blocked 74+/-8% of the total HVA Ca(2+) currents of cerebellar granule neurones, without affecting the low-voltage activated (LVA) current. P/Q/R-type currents in cerebellar granule neurones, isolated using 4 microM nicardipine and 100 nM omega-conotoxin GVIA, were markedly (79+/-6%) inhibited by 60 nM omega-PnTx3-3. R-type currents, isolated either by additional application of 0.5-1 microM of omega-agatoxin IVA or by pre-incubation with 5 microM omega-conotoxin MVIIC were inhibited almost totally by 120 nM of omega-PnTx3-3. omega-PnTx3-3 reversibly altered the kinetics of the P/Q/R current, increasing the degree of inactivation that occurred during a 50 ms pulse from 20% to 40%. N-type currents, recorded from neuroblastoma N18 cells, were partially (34+/-2%) inhibited by 320 nM omega-PnTx3-3. L-type currents, recorded from GH3 cells, were partially (45+/-12%) inhibited by 80 nM omega-PnTx3-3. We conclude that omega-PnTx3-3 inhibits all known HVA Ca(2+) channels, and most effectively the P/Q- and R-type currents.

Electrophysiological characterization and molecular identification of the Phoneutria nigriventer peptide toxin PnTx2-61

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2‐6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate‐rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2‐6. The purified toxin exerted complex effects on Na+ current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na+ conductance and the steady‐state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K D of 0.8 μM for the toxin–channel complex.

Linalool blocks excitability in peripheral nerves and voltage-dependent Na+ current in dissociated dorsal root ganglia neurons

Linalool is a terpene that occurs as a major constituent of essential oils of many plants of widespread distribution. It possesses several biological and pharmacological activities, including depressant effects on the central nervous system and olfactory receptors. The present study investigated whether linalool affects the excitability of peripheral components of the somatic sensory system. We used sciatic nerve and preparations of intact and dissociated neurons of dorsal root ganglion for extracellular, intracellular and patch-clamp recordings. Linalool concentration-dependently (0.3-2.0mM) and reversibly blocked the excitability of the sciatic nerve. It inhibited peak-to-peak amplitude of the compound action potential (IC(50) was 0.78+/-0.04 mM). At 0.8mM, it reversibly increased rheobase and chronaxy (from 3.2+/-0.1 V and 52.4+/-4.1 micros to 4.2+/-0.3 V and 71.2+/-5.5 micros (n=5), respectively) and inhibited with greater pharmacological potency the amplitude of the compound action potential components corresponding to axons with slower velocity of conduction. In a similar concentration range (0.1-6mM), linalool concentration-dependently and reversibly blocked the generation of action potentials of intact dorsal root ganglion neurons without alteration of resting membrane potential and input resistance, and inhibited the voltage-gated Na(+) current of dissociated dorsal root ganglion neurons. In conclusion, we demonstrated that linalool acts on the somatic sensory system with local anesthetic properties, since it blocked the action potential by acting on voltage-dependent Na(+) channels. This finding is important in showing the potential usefulness of linalool as a pharmacotherapeutic agent.