Esther García-Palomero

Predictive Value of Nuclear Factor κB Activity and Plasma Cytokine Levels in Patients with Sepsis

ABSTRACT The relationship between fluctuating cytokine concentrations in plasma and the outcome of sepsis is complex. We postulated that early measurement of the activation of nuclear factor κB (NF-κB), a transcriptional regulatory protein involved in proinflammatory cytokine expression, may help to predict the outcome of sepsis. We determined NF-κB activation in peripheral blood mononuclear cells of 34 patients with severe sepsis (23 survivors and 11 nonsurvivors) and serial concentrations of inflammatory cytokines (interleukin-6, interleukin-1, and tumor necrosis factor) and various endogenous antagonists in plasma. NF-κB activity was significantly higher in nonsurvivors and correlated strongly with the severity of illness (APACHE II score), although neither was related to the cytokine levels. Apart from NF-κB activity, the interleukin-1 receptor antagonist was the only cytokine tested whose level in plasma was of value in predicting mortality by logistic regression analysis. These results underscore the prognostic value of early measurement of NF-κB activity in patients with severe sepsis.

Tacrine–Melatonin Hybrids as Multifunctional Agents for Alzheimer's Disease, with Cholinergic, Antioxidant, and Neuroprotective Properties

Tacrine–melatonin hybrids are potential multifunctional drugs for Alzheimers disease that may simultaneously palliate intellectual deficits and protect the brain against both β‐amyloid peptide and oxidative stress. Molecular modeling studies show that they target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. They are nontoxic and may be able to penetrate the CNS, according to in vitro PAMPA‐BBB assays.

Greater diversity than previously thought of chromaffin cell Ca2+ channels, derived from mRNA identification studies

Using reverse transcription followed by PCR amplification (RT‐PCR), we have identified multiple messenger RNAs encoding for the neuronal pore‐forming Ca2+ channel subunits α1A (P/Q channel), α1B (N channel), α1D (neuronal/endocrine L channel), α1E (R channel), α1G‐H (T channel) and α1S (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary β2, β3, β4, α2/δ and γ2 subunits were also identified. In agreement with these molecular data, perforated patch‐clamp recordings of whole‐cell Ca2+ currents reveal the existence of functional R‐type Ca2+ channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca2+ channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.

Differential blockade of rat α3β4 and α7 neuronal nicotinic receptors by ω-conotoxin MVIIC, ω-conotoxin GVIA and diltiazem

Rat α3β4 or α7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non‐toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 μM) applied at regular intervals. The N/P/Q‐type Ca2+ channel blocker ω‐conotoxin MVIIC inhibited α3β4 currents with an IC50 of 1.3 μM; the blockade was non‐competitive and reversible. The α7 currents were unaffected. At 1 μM, ω‐conotoxin GVIA (N‐type Ca2+ channel blocker) inhibited by 24 and 20% α3β4 and α7 currents, respectively. At 1 μM, ω‐agatoxin IVA (a P/Q‐type Ca2+ channel blocker) did not affect α7 currents and inhibited α3β4 currents by only 15%. L‐type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on α3β4 nicotinic AChRs. The mechanism of α3β4 currents blockade by ω‐conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage‐dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while ω‐toxins did not. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.

Differential expression of calcium channel subtypes in the bovine adrenal medulla

This study aimed at determining the distribution and expression levels of different subtypes of Ca2+ channels in the bovine adrenal medulla, and whether individual subtypes were more abundant in chromaffin cells exhibiting an adrenergic or a noradrenergic phenotype. In situ hybridization using riboprobes specific for the pore-forming Ca2+ channel α1D (L-type channel), α1B (N-type channel), and α1A (P/Q-type channel) subunits of bovine chromaffin cells showed a broad distribution of the three transcripts in adrenal medulla tissue. However, a tissue-specific expression pattern of individual subunits was found; whereas α1B mRNA was homogeneously distributed throughout the medulla, α1D and α1A transcripts were present at higher densities in the internal medullary area, far away from the adrenal cortex. These results were corroborated by comparative analysis of the α1B, α1D, and α1A products amplified by RT-PCR from total RNA extracted from small pieces of tissue dissected out from external or internal medullary areas. Interestingly, immunohistochemical experiments performed in adrenal gland sections, using antidopamine-β-hydroxylase and anti-phenylethanolamine-N-methyltransferase antibodies, indicated a higher density of noradrenergic over adrenergic chromaffin cells in the internal medullary region. These results provide direct evidence in favor of a heterogeneous distribution of Ca2+ channel subtypes in the adrenal medulla, in agreement with previous functional data showing that blockade of the high K+-elicited responses by dihydropyridines was greater in noradrenergic than in adrenergic chromaffin cells. These differences may be relevant for the differential release regulation of each catecholamine under physiological and pathophysiological conditions.

Serotonergic effects of dotarizine in coronary artery and in oocytes expressing 5-HT2 receptors

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.

Multiple Calcium Pathways Induce the Expression of SNAP-25 Protein in Chromaffin Cells

Abstract: Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24‐48 h enhanced 2.5 to five times the expression of SNAP‐25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L‐type Ca2+ channel blocker) but was unaffected by either ω‐conotoxin GVIA (an N‐type Ca2+ channel blocker) or ω‐agatoxin IVA (a P/Q‐type Ca2+ channel blocker). Combined blockade of N and P/Q channels with ω‐conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of furnidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP‐25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]i), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L‐type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo‐1‐loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.

Capacitative Ca2+ entry into Xenopus oocytes is sensitive to ω-conotoxins GVIA, MVIIA and MVIIC

Abstract We have studied capacitative Ca2+ entry into Xenopus oocytes by depleting intracellular Ca2+ stores with inositol 1,4,5-trisphosphate or thapsigargin. Capacitative Ca2+ entry was evoked by hyperpolarisation and monitored via the Ca2+-activated CI− current. Hyperpolarisation-evoked currents increased with extracellular [Ca2+] in the range 0.9-5 mM, and were reversibly inhibited by extracellular Mg2+ (0.1–10 μM) by up to 60%. Currents were decreased by the voltage-gated Ca2+ channel antagonists ω-conotoxin GVIA, MVIIA and MVIIC (0.3–10 μM) and the inhibition of Ca2+ entry in individual oocytes by ω-conotoxins GVIA and MVIIA was highly heterogeneous, but not additive. Flunarizine (10 μM) and the imidazoles SK&F 96365 (10 μM), miconazole (40 μM) and econazole (40 μM) partly blocked Ca2+ entry. Ca2+ entry was unaffected by calciseptine (300 nM) or α-bungarotoxin (1 μM). The possibility that these compounds might inhibit the Ca2+-activated CI− current rather than capacitative Ca2+ entry itself was examined by recording the CI− current activated by the increase in [Ca2+]i activated by the flash photolysis of caged Ca2+. Eicosatetraynoic acid (2–10 μM) markedly inhibited, and La3+ (1 mM but not 100 μM) potentiated the increase in Ca2+ activated CI− current. In contrast, ω-conotoxins and Mg2+ had no effect on the Ca2+-activated CI− current itself. These findings support the hypothesis that capacitative Ca2+ entry into Xenopus oocytes occurs through channels with a pharmacology similar to that of neuronal non-L type voltage-gated Ca2+ channels.

Calcium Channels for Exocytosis in Chromaffin Cells

Publisher Summary The subsequent availability of powerful pharmacological tools allowed the separation of various subcomponents in the whole-cell currents through neuronal Ca 2+ channels. Earlier results led to the conclusion that bovine chromaffin cells expressed L-type Ca 2- channels (15-20%), N-type channels (30-40%), as well as P-type channels (30-40%). It has been found that the divalent cation concentration strongly modified the ability of w-toxins to block specific Ca 2- channel subtypes; this was particularly relevant for ω-conotoxin MVIIC, whose blockade was markedly slowed down and decreased in the presence of 10-mM Ba 2+ , as compared with 5- or 2-mM Ba 2+ . Not all Ca 2+ channel subtypes of bovine chromaffin cells participate equally in the control of the delivery of external Ca 2+ to the secretory machinery to trigger a secretory response on cell depolarization. Thus catecholamine release triggered by brief pulses of high-K+ solutions in superfused cells, measured online with an electrochemical detector, was partially inhibited by 3- μ M furnidipine, by 2- μ M ω-agatoxin IVA, or by 3- μ M ω-conotoxin MVIIC. For long time, no functional role was found for materials coreleased with adrenaline and noradrenaline during exocytosis, that is, ATP and the opiate peptide methionine-enkephalin. Recent findings suggest that they may regulate the kinetics of Ca 2+ channels in chromaffin cells in two ways: slowing down of the current activation and inhibition of the current amplitude.