Angélica Almanza

Calcium current in type I hair cells isolated from the semicircular canal crista ampullaris of the rat.

The low voltage gain in type I hair cells implies that neurotransmitter release at their afferent synapse should be mediated by low voltage activated calcium channels, or that some peculiar mechanism should be operating in this synapse. With the patch clamp technique, we studied the characteristics of the Ca(2+) current in type I hair cells enzymatically dissociated from rat semicircular canal crista ampullaris. Calcium current in type I hair cells exhibited a slow inactivation (during 2-s depolarizing steps), was sensitive to nimodipine and was blocked by Cd(2+) and Ni(2+). This current was activated at potentials above -60 mV, had a mean half maximal activation of -36 mV, and exhibited no steady-state inactivation at holding potentials between -100 and -60 mV. This data led us to conclude that hair cell Ca(2+) current is most likely of the L type. Thus, other mechanisms participating in neurotransmitter release such as K(+) accumulation in the synaptic cleft, modulation of K(+) currents by nitric oxide, participation of a Na(+) current and possible metabotropic cascades activated by depolarization should be considered.

Recombinant human ZP3-induced sperm acrosome reaction: evidence for the involvement of T- and L-type voltage-gated calcium channels.

For successful fertilization mammalian spermatozoa must undergo the acrosome reaction (AR), an exocytotic event that allows this cell to penetrate the outer layer of the oocyte, the zona pellucida (ZP). Four glycoproteins (ZP1-ZP4) compose the human ZP, being ZP3 the physiological inductor of the AR. This process requires changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) involving not fully understood mechanisms. Even in mouse sperm, the pharmacologically documented participation of voltage-gated Ca(2+) (Ca(V)) channels and store-operated channels (SOCs) in the ZP-induced AR is being debated. The situation in human sperm is even less clear due to the limited availability of human ZP. Here, we used recombinant human ZP3 (rhZP3) produced in baculovirus-infected Sf9 cells to investigate the involvement of Ca(V) channels in the human sperm AR. Our findings showed that Ni(2+) and mibefradil at concentrations that block T-type or Ca(V)3 channels, and nimodipine and diltiazem that block L-type or Ca(V)1 channels, significantly inhibited the rhZP3-initiated AR. On the other hand, the AR was insensitive to concentrations of omega-Agatoxin IVA, omega-Conotoxin GVIA and SNX-482 that block P/Q, N and R-type channels, respectively (Ca(V)2 channels). Our overall findings suggest that Ca(V)1 and Ca(V)3 channels participate in human sperm AR. Consistent with this, we detected in human sperm transcripts for the Ca(V)1 auxiliary subunits, alpha(2)delta, beta(1), beta(2) and beta(4), but not the neuronal specific isoforms beta(3) and gamma(2).

Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization- activated current in vestibular ganglion neurons

Properties, developmental regulation, and cAMP modulation of the hyperpolarization-activated current (I(h)) were investigated by the whole cell patch-clamp technique in vestibular ganglion neurons of the rat at two postnatal stages (P7-10 and P25-28). In addition, by RT-PCR and immunohistochemistry the identity and distribution of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) isoforms that generate I(h) were investigated. I(h) current density was larger in P25-28 than P7-10 rats, increasing 410% for small cells (<30 pF) and 200% for larger cells (>30 pF). The half-maximum activation voltage (V(1/2)) of I(h) was -102 mV in P7-10 rats and in P25-28 rats shifted 7 mV toward positive voltages. At both ages, intracellular cAMP increased I(h) current density, decreased its activation time constant (τ), and resulted in a rightward shift of V(1/2) by 9 mV. Perfusion of 8-BrcAMP increased I(h) amplitude and speed up its activation kinetics. I(h) was blocked by Cs(+), zatebradine, and ZD7288. As expected, these drugs also reduced the voltage sag caused with hyperpolarizing pulses and prevented the postpulse action potential generation without changes in the resting potential. RT-PCR analysis showed that HCN1 and HCN2 subunits were predominantly amplified in vestibular ganglia and end organs and HCN3 and HCN4 to a lesser extent. Immunohistochemistry showed that the four HCN subunits were differentially expressed (HCN1 > HCN2 > HCN3 ≥ HCN4) in ganglion slices and in cultured neurons at both P7-10 and P25-28 stages. Developmental changes shifted V(1/2) of I(h) closer to the resting membrane potential, increasing its functional role. Modulation of I(h) by cAMP-mediated signaling pathway constitutes a potentially relevant control mechanism for the modulation of afferent neuron discharge.

Insulin-mediated upregulation of T-type Ca2+ currents in GH3 cells is mediated by increased endosomal recycling and incorporation of surface membrane Cav3.1 channels.

Growth factors and hormones have both short- and long-term regulatory effects on the functional expression of voltage gated Ca2+ (CaV) channels. In particular, it has been reported that chronic treatment with insulin upregulates T-type channel membrane expression, leading to an increase in current density in clonal pituitary GH3 cells. Though this regulatory action may result from alterations in gene expression, recent studies have demonstrated also that endosomal trafficking provides a mechanism for dynamic changes in CaV channel membrane density. Therefore, in the present work we sought to determine whether the actions of insulin on T-type channel functional expression are mediated by transcriptional and/or post-transcriptional mechanisms. Using real-time RT-PCR and semi-quantitative western blot we found no changes after treatment in the transcript and protein levels of Cav3.1, the T-type channel isoform preferentially expressed in the GH3 cells. Consistent with this, transcriptional studies using a luciferase reporter assay suggested that insulin treatment does not affect the Cav3.1 promoter activity. In contrast, patch-clamp recordings on HEK-293 cells stably expressing Cav3.1 channels showed a significant increase in current density after treatment, suggesting that the effects of insulin may require post-transcriptional regulation. In line with this, disruption of the endosomal recycling pathway using Brefeldin A and a dominant negative mutant of the small GTPase Rab11a prevented the stimulatory effects of insulin on Cav3.1 channels in HEK-293 cells. These results may help explain the effects of insulin on T-type channels and contribute to our understanding of how endosomal recycling impacts the functional expression of CaV channels.

Nitric oxide in the amphibian (Ambystoma tigrinum) lateral line

Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum) lateral line. Hair cells of neuromast organs of the head skin and neurons of the postotic ganglia showed a significant NADPH-d reaction. Multiunit recording of neuromast afferent activity was also performed. Nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) produced an initial slight excitation followed by a significant inhibition of the resting discharge of neuromast afferent neurons. In contrast N(G)-nitro-L-arginine (L-NOARG) produced non-significant actions on the afferent neurons discharge. These findings suggest that afferent neurons and hair cells of the lateral line produce nitric oxide that plays an active role in the mechanisms sustaining basal spike discharge in afferent neurons.

pH modulates the vestibular afferent discharge and its response to excitatory amino acids.

&NA; In the isolated inner ear of the axolotl (Ambystoma tigrinum) acid pH decreased and basic pH increased the resting and mechanically evoked spike discharge of semicircular canal afferent neurons. Variations in pH also modified the afferent neuron response to N‐methyl‐D‐aspartic acid (NMDA) acid and to (±)‐&agr;‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA). Responses to both excitatory amino acid agonists increased at pH 7.8 (41% and 22%, respectively) and decreased by perfusion of the preparation with a saline solution, of pH 7.0 (28% in both cases). These results indicate that vestibular endorgans have a significant sensitivity to pH that could play a significant role in various pathological states, and may also contribute to the post‐transductional processing of sensory information.

Inhibition of Peripheral Nociceptors by Aminoglycosides Produces Analgesia in Inflammatory Pain Models in the Rat

Aminoglycosides (AGs) modulate nociceptors and ionic channels expressed in sensory neurons. The AG applied in situ could be useful to alleviate hyperalgesia in animal models of inflammatory pain. We tested streptomycin (ST) and neomycin (NEO) as analgesic agents applied in situ in rat paw inflammation caused by formalin or carrageenan administration. The action of ST and NEO on the action potential discharge produced by acidic stimuli in isolated dorsal root ganglion neurons was also studied in current-clamp recordings. In the formalin test, ST and NEO significantly reduced the nociceptive behaviour. ST reduced the N-(4-methyl-2-quinazolinyl)-guanidine (GMQ)-induced nociceptive behaviour, and NEO diminished the hyperalgesia to thermonociception and mechanonociception produced by CAR. In the current-clamp experiments, ST and NEO reduced the generation of action potentials when an acidic solution was applied. We conclude that ST and NEO produce analgesia to inflammatory pain, an effect that is due in part to the inhibition of ASIC activation in sensory neurons.

A D2-like receptor family agonist produces analgesia in mechanonociception but not in thermonociception at the spinal cord level in rats

The administration of dopaminergic drugs produces analgesia in individuals experiencing different types of pain. Analgesia induced by these drugs at the spinal cord level is mediated by D2-like agonists, which specifically inhibit the detection of nociceptive stimuli by sensory afferents. The extent of the analgesia provided by spinal dopamine agonists remains controversial, and the cellular mechanism of this analgesic process is poorly understood. The objective of this study was to evaluate the analgesic effect of quinpirole, a D2-like agonist, based on two nociceptive tests and at various doses that were selected to specifically activate dopamine receptors. We found that intrathecal quinpirole administration produces analgesia of mechanical but not thermal nociception and that the analgesic effect of quinpirole is reversed by a mix of D2, D3, and D4 receptor-specific antagonists, suggesting that the activation of all D2-like receptors is involved in the analgesia produced by intrathecal quinpirole. The differential effect on thermal and mechanical nociception was also tested upon the activation of μ-opioid receptors. As reported previously, low doses of the μ-opioid receptor agonist DAMGO produced analgesia of only thermonociception. This evidence shows that a D2-like receptor agonist administered at the spinal cord level produces analgesia specific to mechanonociception but not thermonociception.

Upregulation of voltage-gated Ca2+ channels in mouse astrocytes infected with Theiler’s murine encephalomyelitis virus (TMEV)

Theilers murine encephalomyelitis virus (TMEV) induces demyelination in susceptible strains of mice through a CD4(+) Th1 T cell-mediated immunopathological process. TMEV infection produces a syndrome in mice that resembles multiple sclerosis. In this work, we focused on the increased expression of the genes encoding voltage-gated Ca(2+) channel subunits in SJL/J mouse astrocytes infected in culture with a BeAn strain of TMEV. Affymetrix DNA murine genome U74v2 DNA microarray hybridized with cRNA from mock- and TMEV-infected astrocytes revealed the upregulation of four sequences encoding Ca(2+)-binding and Ca(2+) channel subunit proteins. The DNA hybridization results were further validated using conventional RT-PCR and quantitative RT-PCR, demonstrating the increased expression of mRNA encoding channel subunit proteins. Western blotting also showed the increased synthesis of L- and N-type channel subunit specific proteins after infection. The reduced expression and the functional upregulation of functional voltage-gated Ca(2+) channels in mock- and TMEV-infected cells, respectively, was demonstrated using voltage clamp experiments. TMEV infection in mouse astrocytes induced a Ca(2+) current with a density proportional to the amount of viral particles used for infection. The use of Ca(2+) channel blockers, nimodipine and ω-conotoxin-GVIA, showed that both functional L- and N-type Ca(2+) channels were upregulated in infected astrocytes. The upregulation of Ca(2+) channels in astrocytes after TMEV infection provides insight into the molecular processes and potential role of astrocyte Ca(2+) dysregulation in the pathophysiology of encephalomyelitis and is important for the development of novel therapeutic strategies leading to prevention of neurodegeneration.

FMRFamide-related peptide expression in the vestibular-afferent neurons.

Vestibular-afferent neurons innervate hair cells from the sensory epithelia of vestibular end-organs and their action-potential discharge dynamics are driven by linear and angular accelerations of the head. The electrical activity of the vestibular-afferent neurons depends on their intrinsic properties and on the synaptic input from hair cells and from the terminals of the efferent system. Here we report that vestibular-afferent neurons of the rat are immunoreactive to RFamide-related peptides, and that the stronger signal comes from calyx-shaped neuron dendrites, with no signal detected in hair cells or supporting cells. The whole-cell voltage clamp recording of isolated afferent neurons showed that they express robust acid-sensing ionic currents (ASICs). Extracellular multiunit recordings of the vestibular nerve in a preparation in vitro of the rat inner ear showed that the perfusion of FMRFamide (a snail ortholog of this family of neuropeptides) exerts an excitatory effect on the afferent-neurons spike-discharge rate. Because the FMRFamide cannot activate the ASIC but reduces its desensitization generating a more robust current, its effect indicates that the ASIC are tonically active in the vestibular-afferent neurons and modulated by RFamide-like peptides.