Descripción: In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both α9 and α10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that α9 mRNA is expressed in adult IHCs but that α10 mRNA disappears after the onset of hearing. In the present study, antibodies to α10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of α10, but not α9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

Descripción: Studies of the electrophysiological response to acetylcholine (ACh) in mammalian outer hair cells (OHCs) are hindered by the presence of a large potassium current, IK,n, most likely mediated by channels containing the KCNQ4 subunit. Since IK,n can be blocked by linopirdine, cholinergic effects might be better revealed in the presence of this compound. The aim of the present work was to study the effects of linopirdine on the ACh-evoked responses through α9α10-containing native and recombinant nicotinic cholinergic receptors. Responses to ACh were blocked by linopirdine in both OHCs and inner hair cells (IHCs) of rats at postnatal days 21-27 (OHCs) and 9-11 (IHCs). In addition, linopirdine blocked responses of recombinant α9α10 nicotinic cholinergic receptors (nAChRs) in a concentration-dependent manner with an IC50 of 5.2 μM. Block by linopirdine was readily reversible, voltage independent, and surmountable at high concentrations of ACh, thus suggestive of a competitive type of interaction with the receptor. The present results contribute to the pharmacological characterization of α9α10-containing nicotinic receptors and indicate that linopirdine should be used with caution when analyzing the cholinergic sensitivity of cochlear hair cells.

Descripción: The Maastrichtian-Early Palaeocene Salamanca Formation and equivalent marine sequences show several glaucony levels, which could be used as a source of potassium and could partially replace current fertilizer requirements met from import. First agronomic assays are shown comparing the efficiency of greensands (GL) with a traditional potassium fertilizer (KCl). Prospection and sampling took place in the heads of Rio Chico to east of Lago Colhué Huapi, Chubut, where glauconitic levels thickness reach to 20m. This research was separated in phases: phase 1, prospection, sampling, and sedimentological and chemical routine analyses, phase 2a, concentration and ore treatment assays, and phase 2b, agronomic assays. This last phase, was done in culture chamber, with 125 μm and 250 μm fractions (2,85 and 2,82 % K 2 O respectively) and KCl. Varying the dose, seven treatments were completed. In successive harvests (four in total) was observed that GL1 and GL2 (125), did not present a significantly differential behavior with the control. Both treatments of GL 250 μm showed major accumulated biomass, overcoming the control in 44 and 60 % respectively. As conclusion, better results were obtained by 250 μm overcoming even to the KCl fertilization. Probably the major participation of K feldspars in the 125 μm and fast leaching of K in this fraction during successive irrigations of soil would originate the minor observed yield. The great areal extension of the Salamanca Formation and the continuity of the glauconitic levels encourage continuing the evaluation as fertilizer or amendment.

Descripción: The crude polysaccharide from cystocarpic Callophyllis variegata was fractionated with potassium chloride yielding three minor fractions which precipitated between 0.05-0.10 M KCl, 1.20-1.25 M KCl and 1.80-2.00 M KCl, and a main product soluble in 2.00 M KCl. These fractions were analysed and structural analysis of the major one was carried out by methylation, FT-IR and 13C NMR.

Descripción: The polysaccharides from cystocarpic Iridaea undulosa, soluble and insoluble in 2M potassium chloride, Cs and Ci, respectively, were treated with alkali and fractionated by precipitation with increasing concentrations of KCl. They were later separated by ion-exchange chromatography, to yield fractions enriched in an α-(1→6)-glucan, agaroids and carrageenans.

Descripción: In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic α9α10 receptor coupled to the activation of SK2 Ca 2+-activated K+ channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q- and N-type voltage-gated calcium channels (VGCCs), ω-agatoxin IVA and ω-conotoxin GVIA, respectively, we show that Ca2+ entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca2+ entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca2+-activated K+ channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons. Copyright © 2010 the authors.

Descripción: Efferent inhibition of cochlear hair cells is mediated by α9α10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the α10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 α10 transgenic mice backcrossed to a Chrna10 -/- background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the α10 transgene restored nAChR function. However, in the α10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh. © 2009 Association for Research in Otolaryngology.

Descripción: The existence of ammonium anomalies in mineralized deposits in Argentina is presented. These anomalies are given by the substitution of potassium ion by ammonium ion in certain feldspars, clay minerals and sulfates. This substitution doesn't produce physical changes either in the minerals or the host rocks. Ammonium-bearing minerals have been found in several hydrothermally altered metallic deposits. Ammonium ion is specially sensible to SWIR reflectance spectroscopy method, so it became an ideal, easy and valuable tool for ammonium anomalies detection. Although ammonium-bearing minerals are not always associated with metallic ore, they can be used as an exploration guide, being thus a very useful prospective method for metallic deposits in general. © 2006 Asociación Geológica Argentina.

Descripción: 1. 1. By chromatography through Sephadex G-200 of increasing amounts of partially purified pig liver ALA-D, different profiles were obtained, showing that species of molecular weights ranging from 140,000 to 560,000 might exist in equilibrium, but their relative ratio was dependent on the total amount of protein sampled. In all cases, however, the main peak (60-70%) corresponded to the 280,000 MW oligomer, that is the octamer. 2. 2. It was found that elution profiles were also dependent on column dimensions and on the purity of the enzyme preparation. 3. 3. K+ ions affected both catalytic activity and aggregation of the enzyme. 4. Results here reported add further support to the proposal of the existence of a minimal functional dimer. © 1980.

Descripción: 1. The effects of the voltage-dependent calcium channel (VDCC) blockers ω-agatoxin IVA (ω-AgaIVA), ω-conotoxin GVIA (ω-CgTx), ω-conotoxin MVIIC (ω-MVIIC) and ω-conotoxin MVIID (ω-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of ω-AgaIVA and ω-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, I(ca), and I(K(Ca)), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker ω-AgaIVA (100 nM) and P- Q- and N-type channel blockers ω-MVIIC (1 μM) and ω-MVIID (3 μM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker ω-CgTx (5 μM) was ineffective. 3. The process of release was much more sensitive to ω-MVIIC (IC50 = 39 nM) than to ω-MVIID (IC50 = 1.4 μM). After almost completely blocking transmitter release (quantal content ~0.3% of its control value) with 3 μM ω-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of ~20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 ± 0.04 to 4.8 ± 1.4). 4. Nerve-evoked transmitter release in a low Ca2+-high Mg2+ medium (low release probability, quantal content = 2 ± 0.1) had the same sensitivity to ω-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K+-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of ω-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33°C. Conversely, the effect of ω-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, I(ca), and I(K(Ca)), respectively, were highly sensitive to low concentrations (10-30 nM) of ω-AgaIVA. The I(ca), and the I(K(Ca)) were also strongly reduced by 1 μM ω-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with ω-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.

Descripción: Bath application of compound T-588, a neuroprotective agent, reduced paired-pulse and repetitive-pulse facilitation at mammalian and crustacean neuromuscular junctions. In addition, it reduced voltage-gated sodium and potassium currents in a use-dependent fashion, but had only a small effect on the presynaptic Ca 2+ conductance. By contrast, it blocked FM 1-43 vesicular uptake but not its release, in both species. Postsynaptically, T-588 reduced acetylcholine currents at the mammalian junction in a voltage-independent manner, but had no effect on the crayfish glutamate junction. All of these effects were rapidly reversible and were observed at concentrations close to the compound's acute protective level. We propose that this set of mechanisms, which reduces high-frequency synaptic transmission, is an important contributory factor in the neuroprotective action of T-588.

Descripción: Cerro Falkner is composed of granitic rocks underlying volcanic rocks of the Ventana Formation of Paleogene age. Those intrusives are graniodioritic in composition, metaluminous to peraluminous, subalkaline, with characteristics of volcanic arc granites that are concordant with other granitoids of the Cordillera Patagónica Septentrional's northern segment. The age obtained by the K-Ar method on tschemakitic amphiboles (125 ± 20 Ma) points out a minimun age from the lower Cretaceous for the Cerro Falkner's granodiorite. Therefore the use of the term Los Machis Formation (Upper Cretaceous) would be incorrect for these rocks. This age, allows us to relate the granitic rocks on the study area to the lower Cretaceous, coeval with the volcanism of the Divisadero Group.

Descripción: The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the α9α10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the α9α10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 μM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 μM ryanodine. This facilitatory effect on current through the AChR could enhance brief (∼1 s) activation of associated calcium-dependent K+ (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate α9α10- mediated responses and for potential inner ear therapeutics based on this interaction. © 2007 Association for Research in Otolaryngology.

Descripción: Transmission at the mouse neuromuscular junction normally relies on P/Q-type channels, but became jointly dependent on both N-and R-type Ca2+ channels when the P/Q-type channel α1A subunit was deleted. R-type channels lay close to Ca2+ sensors for exocytosis and IK(Ca) channel activation, like the P/Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca2+ channels in the hierarchy P/Q>R>N. The α1A-/- neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca2+/Mg2+ ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters.

Descripción: The 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11βHSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11βHSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 ± 3.1 nM, and that for NAD+ was approximately 8 μM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity, 11α-Hydroxyprogesterone (11αOH-P) was an order of magnitude more potent a competitive inhibitor of the 11βHSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 0.9 vs. 15 nM). 11βOH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5α-pregnandione and 5β-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11αOH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11αOH-P was not metabolized by 11βHSD-2. We were unable to demonstrate the presence of 11αOH-P in human urine. In conclusion, a cell line stably transfected with the rat 11βHSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11αOH-P was found to be a potent relatively specific inhibitor of the 11βHSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11βHSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme.

Descripción: In the mammalian inner ear, the gain control of auditory inputs is exerted by medial olivocochlear (MOC) neurons that innervate cochlear outer hair cells (OHCs). OHCs mechanically amplify the incoming sound waves by virtue of their electromotile properties while the MOC system reduces the gain of auditory inputs by inhibiting OHC function. How this process is orchestrated at the synaptic level remains unknown. In the present study, MOC firing was evoked by electrical stimulation in an isolated mouse cochlear preparation, while OHCs postsynaptic responses were monitored by whole-cell recordings. These recordings confirmed that electrically evoked IPSCs (eIPSCs) are mediated solely by α9β10 nAChRs functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1 Hz. However, as the stimulation frequency was raised, the reliability of release increased due to presynaptic facilitation. In addition, the relatively slow decay of eIPSCs gave rise to temporal summation at stimulation frequencies >10 Hz. The combined effect of facilitation and summation resulted in a frequency-dependent increase in the average amplitude of inhibitory currents in OHCs. Thus, we have demonstrated that short-term plasticity is responsible for shaping MOC inhibition and, therefore, encodes the transfer function from efferent firing frequency to the gain of the cochlear amplifier. © 2011 the authors.

Descripción: The effects of dopamine (DA) and dopaminergic agonists and antagonists on ion transport were studied in isolated perfused gills of the crab Chasmagnathus granulatus. DA applied under steady state conditions (perfusion with hemolymph-like saline) produced a transient increase of the transepithelial potential difference (Vtc) from 2.2±0.2 to 4.8±0.3 mV, describing an initial cAMP-dependent stimulating phase followed by an inhibitory phase. Spiperone and domperidone (antagonists of D2-like DA receptors in vertebrates) completely blocked the response to DA, while the D1-like antagonist SCH23390 blocked only the inhibitory phase. Theophylline (phosphodiesterase inhibitor) and okadaic acid (protein phosphatases PP1 and PP2A inhibitor) were also able to block the inhibitory phase, suggesting that it depends on adenylyl cyclase inhibition and on protein phosphatases. When the gills were perfused with hypoosmotic solution, or with the adenylyl cyclase activator forskolin, Vte was increased several-fold. DA applied under these stimulated conditions partially reversed the Vte increase by 54% and 25%, respectively. Similarly, the D1-like agonist, fenoldopam, produced a 33% reduction in the stimulated Vte. We propose that, in C. granulatus gills, DA stimulates adenylyl cyclase and therefore ion transport through D1-like receptors linked to a Gs protein, although they respond to antagonists that interact with D2-like receptors in vertebrates. The inhibitory phase seems to be mediated by D2-like receptors linked to a Gi/o protein, which inhibits adenylyl cyclase, although these receptors can be activated or blocked by agonists or antagonists that interact with D1-like receptors in vertebrates and insects.

Descripción: We studied the transepithelial potential difference (TEPD) and 22Na flux across isolated perfused gills (anterior pair 5 and posterior pairs 6-8) of the crab Chasmagnathus granulatus acclimated to either hypo- or hyper-osmotic conditions. The gills of crabs acclimated to low salinity, perfused and bathed with 10%‰ saline solutions, produced the following TEPDs (hemolymph side with respect to bath side): 0.4±0.7, -10.2±1.6, -10.8±1.3 and -6.7±1.3mV for gills 5, 6, 7 and 8, respectively. Gills 6, 7 and 8 did not differ significantly. Reducing the saline concentration of bath and perfusate from 30%‰ to 20%‰ or 10%‰ increased significantly the TEPDs of these gills. TEPDs of gill 6 (representative of posterior gills) were reduced by 69±5% and 60±5% after perfusion with ouabain or BaCl2 (5mmoll-1 each), respectively. The same gill showed a net ouabain-sensitive Na+ influx of 1150±290 μequiv g-1h-1. Gill 6 of crabs acclimated to high salinity produced TEPDs of -1.5±0.1 and -1.3±0.09mV after perfusion with 30%‰ or 40%‰ salines, respectively. Perfusion with ouabain or BaCl2 reduced TEPDs by 76±7% and 86±4%, respectively. A net ouabain-sensitive Na+ efflux of 2282±337 μequiv g-1h-1 was recorded in gill 6 perfused with 38%‰ saline.

Descripción: It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca2+ channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca2+ current. Accordingly, in the present work we found that the Ca2+ current flowing through P/Q-type Ca2+ channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca2+ current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K+ stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca2+ channels. © 2013 Álvarez et al.