Phenotype in patients with intellectual disability and pathological results in array CGH. / Fenotipo en pacientes con discapacidad intelectual y array-CGH patológico.

INTRODUCTION: Global developmental delay (GDD) and intellectual disability (ID) are frequent reasons for consultation in paediatric neurology departments. Nowadays, array comparative genomic hybridisation (array-CGH) is one of the most widely used techniques for diagnosing these disorders. Our purpose was to determine the phenotypic features associated with pathological results in this genetic test. METHODS: We conducted a blind study of the epidemiological, clinical, anthropometric, and morphological features of 80 patients with unexplained ID to determine which features were associated with pathological results in array-CGH. RESULTS: Pathological results were found in 27.5% of the patients. Factors associated with pathological results in array-CGH were a family history of GDD/ID (OR = 12.1), congenital malformations (OR = 5.33), having more than 3 facial dysmorphic features (OR = 20.9), and hypotonia (OR = 3.25). CONCLUSIONS: Our findings are consistent with those reported by other published series. We therefore conclude that the probability of having pathological results in array-CGH increases with the presence of any of the features mentioned above in patients with ID/GDD.

Effects of whole body vibration training on balance in adolescents with and without Down syndrome.

The present study aimed to determine whether a whole body vibration training program (WBV) is able to improve static standing balance in adolescents with and without Down syndrome (DS). Thirty adolescents with DS aged 11-20 years (DSG) and 27 adolescent, age/sex matched, without DS (CG) joined the study. Participants of each group were divided into two comparable groups, those who performed WVB (in DSG: VDSG; in CG: VCG) and those who did not perform WVB (in DSG: nVDSG; in CG: nVCG). Static-standing-balance under four conditions (C1: open-eyes/fixed-foot-support; C2: closed-eyes/fixed-foot-support; C3: open-eyes/compliant-foot-support; C4: closed-eyes/compliant-foot-support) was examine, before and after a 20-week WBV training program. For balance study, Postural-Parameters (PPs), based on center of pressure (COP) oscillations (anterior/posterior and medial/lateral COP excursion and COP mean velocity), and PPs ratios among the four conditions were calculated. After WBV training, no significant differences were found in any parameter in the VCG and nVCG and neither in the nVDSG, but there was a decrease of mean values in the analyzed PPs under C4, with significant differences in medial/lateral COP excursion and COP mean velocity, and a significant decrease in the ratio C4/C1 of the mean velocity in VDSG. Therefore, WBV training had positive effects in the balance of DS adolescents although only under specific conditions, with vision and somatosensory input altered. The positive results of this study are encouraging and open a wide field of research, looking for the most efficient program for this population.

SEMICYUC 2012. Recommendations for intensive care management of acute pancreatitis.

OBJECTIVE: Significant changes in the management of acute pancreatitis have taken place since the 2004 Pamplona Consensus Conference. The objective of this conference has been the revision and updating of the Conference recommendations, in order to unify the integral management of potentially severe acute pancreatitis in an ICU. PARTICIPANTS: Spanish and international intensive medicine physicians, radiologists, surgeons, gastroenterologists, emergency care physicians and other physicians involved in the treatment of acute pancreatitis. LEVELS OF EVIDENCE AND GRADES OF RECOMMENDATION: The GRADE method has been used for drawing them up. DRAWING UP THE RECOMMENDATIONS: The selection of the committee members was performed by means of a public announcement. The bibliography has been revised from 2004 to the present day and 16 blocks of questions on acute pancreatitis in a ICU have been drawn up. Firstly, all the questions according to groups have been drawn up in order to prepare one document. This document has been debated and agreed upon by computer at the SEMICYUC Congress and lastly at the Consensus Conference which was held with the sole objective of drawing up these recommendations. CONCLUSIONS: Eighty two recommendations for acute pancreatitis management in an ICU have been presented. Of these 84 recommendations, we would emphasize the new determinants-based classification of acute pancreatitis severity, new surgical techniques and nutritional recommendations. Note. This summary only lists the 84 recommendations of the 16 questions blocks except blocks greater relevance and impact of its novelty or because they modify the current management.

Static standing balance in adolescents with Down syndrome.

AIM: To analyse static-standing-balance of adolescents with Down syndrome (DS). METHODS: Thirty-two adolescents with DS aged 10-19 years (DSG); 33 adolescents, age/sex-matched, without DS (CG). Static-standing-balance under four conditions (C1: open-eyes/fixed-foot-support; C2: closed-eyes/fixed-foot-support; C3: open-eyes/compliant-foot-support; closed-eyes/compliant-foot-support) was examined by means of time and frequency Postural-Parameters (PPs). To evaluate the contribution of each sensory system influencing postural control ratios among the four conditions were calculated. Mean values of all PPs were higher in the DSG than in the CG. Mean values of time PPs were higher in both groups on compliant-foot-support (with open and closed eyes) than on fixed-foot-support. Ratios C2/C1 were significantly lower in DSG than in CG; ratios C3/C1 presented higher values in DSG than in CG, with significant differences in length path and RMS-velocity; there were no differences in ratios C4/C1. CONCLUSIONS: In our group of DS adolescents the shift from visual to multimodal control of stance had occurred and they showed similar postural control patterns than non-DS. Even though, they presented worse static balance than their peers without DS and they had more problems with altered somasosensory input. An adequate rehabilitation program insisting on somatosensory input could be a useful measure to improve balance.

Kinematic assessment of paediatric forefoot varus.

Forefoot varus is a static deformity not easy to assess clinically. If left uncorrected, it is thought to affect both the posture of the patient and the kinematics of their lower limbs, and even the spine. Three-dimensional gait assessment could help to confirm forefoot varus diagnosis and provide objective evidence of the functional adaptive mechanisms postulated in the literature. The recently available Oxford Foot Model was used, simultaneously with a conventional lower limb model, to compare the kinematics of 10 forefoot varus children (aged 8-13) and 11 healthy controls (aged 7-13) during gait. Data acquisition was performed using a six-camera motion capture system, with a total of 27 reflective markers. A patient-by-patient comparison with the controls suggested several compensation patterns, although statistically significant differences were found only for the mean values of hip adduction/abduction during load response and midstance and hip flexion/extension during pre-swing. A multivariate statistical technique was used to determine which of the measured variables better separated both groups. The best discriminant model presented here includes hip adduction/abduction during load response, hindfoot/tibia inversion/eversion during pre-swing, hindfoot/tibia dorsiflexion/plantar flexion during load response and arch height during midstance, providing a rate of correct classification of 81%. The results could not fully confirm the kinematic relationships suggested in the literature. The small degree of forefoot varus deformity present in the patient group could have prevented other variables from becoming discriminant. A larger patient sample would help determine the possible different compensatory patterns to different degrees of forefoot varus.

Assessment of the medial longitudinal arch in children and adolescents with obesity: footprints and radiographic study.

The purpose of this study was to evaluate foot arch types of obese children and adolescents aged 9-16.5 years using both indirect and direct measures. Fifty-eight obese children/adolescents attending the paediatric endocrinology unit of the University Hospital "Lozano Blesa" in Zaragoza were selected as experimental subjects. Fifty-eight gender and age matched, normal-weight children/adolescents were selected as control subjects. To assess the medial longitudinal arch (MLA) height, which is used as a main reference for the diagnosis of flatfoot, footprints from both feet were collected (in both groups) and lateral weight-bearing radiographs of both feet were taken (of 49 of the 58 obese children). Footprint angle (FA) and the Chippaux-Smirak index (CSI) were calculated from the footprints. Talus-first metatarsal (TFMA) and calcaneal inclination angles (CIA) were obtained from lateral feet radiographs. In the normal-weight group, mean values of FA and CSI indicated a normal MLA. In the obese group, morphological flatfoot was identified. Comparison between both groups, by side and gender, showed a decrease of FA (p<0.001) and an increase of CSI (p<0.001) in obese subjects. Mean values of TFMA and CIA in the obese group indicated a lowering of the MLA. Obese children/adolescents between 9 and 16.5 years of age had significantly lower values of FA and higher CSI, related to a lower MLA. Radiographic parameters supported these findings and mean values were associated with a fall of this arch.

Foot structure in overweight and obese children.

OBJECTIVE. To analyse the association between overweight and obesity and foot structure in children older than 9 years of age, whose longitudinal medial arch (MLA) should be practically established. DESIGN, SETTING AND SUBJECT. A cross-sectional study of 245 children (age: 13.22±1.8 years) from four randomly selected schools (Zaragoza, Spain). Body mass index (BMI) was calculated and normal-weight, overweight and obese groups were defined. Footprints for both feet were collected. MEASUREMENTS. Foot angle (FA) and Chippaux-Smirak index (CSI) were calculated from the footprints. Height and body mass were measured to calculate the BMI. RESULTS. Mean values of FA and CSI in the normal-weight group indicated the presence of a normal MLA; however, in the overweight they indicated an intermediary MLA and in the obese group, a low MLA. Comparison among the groups showed a decrease of FA (p<0.01) and an increase of CSI (p<0.01) with increasing weight associated with a lower MLA. A low but significant correlation (p<0.0001) was found between the z-score BMI and footprint parameters. CONCLUSIONS. In children aged 9 to 16.5 years, the increase of body mass is related to a lower MLA. The MLA is lower in these obese children than in the younger ones studied in previous works probably due to the continuous bearing of excessive mass from childhood. A lower MLA could cause health problems. An assessment of foot structure in these children is recommended as the classification of the foot arch type can help decide if treatment to avoid these problems is necessary.

Bioassay for determining sensitivity to sulfosulfuron on seven plant species.

This study presents a bioassay procedure, based on the root and shoot growth parameters, for the determination of the herbicide sulfosulfuron (1-(4,6 dimethoxypyrimidin-2-yl)-3-(2-ethylsulfonylimidazo[1,2-a]pyridin-3-ylsulfonil)urea) sensitivity on seven vegetal species. Plant response to sulfosulfuron was calculated with the equations fitted to the root growth data as a function of the logarithm of the herbicide concentration by non-linear regression and was used to calculate the doses for 10, 30 and 50% inhibition of root growth (EC10, EC30 and EC50). The results indicate that the phytotoxic effect of sulfosulfuron in all the species assayed followed the order: flax > maize > onion > vetch > lepidium sativum > tomato > barley. These species showed phytotoxicity at low levels of sulfosulfuron and flax appeared to be the most susceptible species to sulfosulfuron (0.001 mg/L).

Residue levels of captan and trichlorfon in field-treated kaki fruits, individual versus composite samples, and after household processing.

The dissipation of residue levels of captan and trichlorfon in field-treated kaki crops was studied according to good laboratory practices to propose maximum residue limits (MRLs). Residue levels of captan and trichlorfon were analysed by GC/MS and LC-MS/MS, respectively. Residue levels of captan and trichlorfon permitted one to propose MRLs in kaki of 3 and 5 mg kg(-1), respectively. The behaviour of these residues was also studied after peeling and cooking, and in individual fruits versus composite samples. Residue levels of these compounds for individual fruits suggested that a variability factor up to three could be set for the acute risk assessment. Levels of captan decreased by more than 90% after peeling and completely after cooking. Trichlorfon penetrates into the flesh in a proportion of 70% of the residue at the pre-harvest interval. Cooking resulted in a decrease of 27% of residue levels of trichlorfon.

New classes of AChE inhibitors with additional pharmacological effects of interest for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is associated to a gradual loss of attention and memory that have been associated to impairment of brain cholinergic neurotransmission, particularly a deficit of cholinergic neurons in the nucleus basalis of Meynert. Thus, it is not surprising that the first therapeutic target that has demonstrated therapeutic efficacy on cognition, behaviour and functional daily activities has been the inhibitors of acetylcholinesterase (AChE), i.e. tacrine, donepezil, rivastigmine and galanthamine. But not all inhibitors of AChE have the same potency to block the enzyme and have a different pharmacological profile. For instance, rivastigmine is a dual inhibitor of AChE and butyrylcholinesterase (BuChE), and galanthamine is a mild inhibitor of AChE and an allosteric potentiating ligand of neuronal nicotinic receptors for acetylcholine (nAChRs). In addition, we have recently found that galanthamine has neuroprotective effects by inducing calcium signals and the induction of the antiapoptotic protein Bcl-2. In this frame, we have been synthesizing new tacrine derivatives that keep their ability to inhibit AChE but that interfere with neuronal calcium overloading and prevent apoptosis. Some of these compounds exhibit neuroprotecting properties and thus, could be useful in the treatment of neurodegenerative and ischaemic brain diseases.

[Familiar chronic progressive external ophthalmoplegia of mitochondrial origin]. / Oftalmoplejía crónica progresiva externa familiar de origen mitocondrial.

INTRODUCTION: The syndrome of chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial disease characterized by ptosis and ophthalmoplegia has that has been associated to the presence of large deletion, single or multiple, in the mitochondrial DNA of skeletal muscle. CASE REPORT: We report a familiar case of chronic progressive external ophthalmoplegia of maternal inheritance that began at birth, and developed with slow progression but with no multisystemic involvement. Non of the affected individuals had ragged-red fibers in skeletal muscle. Genetic analysis of mitochondrial DNA revealed the presence of a single deletion of 4,977 bp that encompasses the nucleotide positions 8,482 to 13,460, flanked by a direct repeat sequence. CONCLUSIONS: The amount of deleted mitochondrial DNA (15%) in this patient's muscle suggests, even if the percentage of the mutation is low, that this deletion is the molecular cause of the phenotypic presentation of this patient. This is one of the few cases described in the literature of CPEO maternally inherited.

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and secretion in bovine chromaffin cells.

Mitochondria play an important role in the homeostasis of intracellular Ca(2+) and regulate its availability for exocytosis. Inhibitors of mitochondria Ca(2+) uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K(+). Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F-actin networks also potentiate the secretory response to high K(+). The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca(2+) from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F-actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F-actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine-rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca(2+) by mitochondria would protect the cell from an enhanced PKC activation and cortical F-actin disassembly, thereby limiting the magnitude of the secretory response.

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death.

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.

Calcium-dependent inhibition of L, N, and P/Q Ca2+ channels in chromaffin cells: role of mitochondria.

The hypothesis that the buffering of Ca(2+) by mitochondria could affect the Ca(2+)-dependent inhibition of voltage-activated Ca(2+) channels, (I(Ca)), was tested in voltage-clamped bovine adrenal chromaffin cells. The protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the blocker of the Ca(2+) uniporter ruthenium red (RR), and a combination of oligomycin plus rotenone were used to interfere with mitochondrial Ca(2+) buffering. In cells dialyzed with an EGTA-free solution, peak I(Ca) generated by 20 msec pulses to 0 or +10 mV, applied at 15 sec intervals, from a holding potential of -80 mV, decayed rapidly after superfusion of cells with 2 microm CCCP (tau = 16.7 +/- 3 sec; n = 8). In cells dialyzed with 14 mm EGTA, CCCP did not provoke I(Ca) loss. Cell dialysis with 4 microm ruthenium red or cell superfusion with oligomycin (3 microm) plus rotenone (4 microm) also accelerated the decay of I(Ca). After treatment with CCCP, decay of N- and P/Q-type Ca(2+) channel currents occurred faster than that of L-type Ca(2+) channel currents. These data are compatible with the idea that the elevation of the bulk cytosolic Ca(2+) concentration, [Ca(2+)](c), causes the inhibition of L- and N- as well as P/Q-type Ca(2+) channels expressed by bovine chromaffin cells. This [Ca(2+)](c) signal appears to be tightly regulated by rapid Ca(2+) uptake into mitochondria. Thus, it is plausible that mitochondria might efficiently regulate the activity of L, N, and P/Q Ca(2+) channels under physiological stimulation conditions of the cell.

Mechanisms of blockade by the novel migraine prophylactic agent, dotarizine, of various brain and peripheral vessel contractility.

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

PF9404C, a new slow NO donor with beta receptor blocking properties.

1. PF9404C is the S-S diesteroisomer of a novel blocker of beta adrenergic receptors with vasodilatory properties. It causes a concentration-dependent relaxation of rat aorta helical strips pre-contracted with 10(-6) M noradrenaline (NA; IC(50) 33 nM). It was equipotent to nitroglycerin (NTG; IC(50) 49 nM), but much more potent than isosorbide dinitrate (ISD; IC(50) 15,000 nM). 2. Oxyhaemoglobin (10 microM) shifted to the right the concentration-response curve for the relaxation induced by PF9404C (IC(50) 530 nM) or NTG (IC(50) 61 nM). 3. Either methylene blue (MB) or ODQ (1 microM each) largely prevented the vasorelaxing responses to increasing concentrations of PF9404C or NTG. 4. In rat aorta smooth muscle cells, PF9404C increased the formation of cyclic GMP from 3 pmol mg(-1) protein in basal conditions, to 53 pmol mg(-1) protein in 10 microM PF9404C. Neither metoprolol nor carvedilol enhanced cyclic GMP. 5. In the electrically driven guinea-pig left atrium, PF9404C blocked the inotropic effects of isoprenaline in a concentration-dependent manner. Its IC(50) (30 nM) was similar to that for S-propranolol (22.4 nM) and lower than the IC(50)s for metoprolol (120 nM) and atenolol (192 nM). The beta-adrenergic ligand (-)-[(3)H]-CGP12177 (0.2 nM) was displaced from its binding to rat brain membranes with K(i) of 7 nM, 17 nM, 170 nM and 1.2 microM respectively for PF9404C, S-(-)propranolol, metoprolol, and atenolol. 6. The data are consistent with the idea that the S-S diesteroisomer PF9404C, is a potent vasorelaxing agent, as well as a blocker of cardiac beta adrenergic receptors. The mechanism of its vasorelaxing effects involves the slow generation of NO. This molecule can, therefore, exhibit antihypertensive and cardioprotective actions through a double mechanism, NO donation and beta blockade.

The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties.

The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP. Moreover, it can form self-assemblies. Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule. Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily. Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane. We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs. Our results demonstrate that 50K is essential for viability depending on the genetic background. We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism.

Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells.

1. In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1.2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0.65 +/- 0.02 fmol cell-1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0.16 +/- 0.01 fmol cell-1. 3. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 +/- 104 nM in hyperpolarized cells to 115 +/- 9 nM in depolarized cells. 4. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1.2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 +/- 35 nA of catecholamines in hyperpolarized cells and 220 +/- 19 nA in depolarized cells. 5. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (2 microM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. 6. This was strengthened by two additional findings: (i) nifedipine (3 microM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 microM), an L-type Ca2+ channel 'activator', dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 microM nifedipine and dramatically enhanced by 3 microM FPL64176. 8. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.