Recurrent pneumothorax after cesarean delivery in the critically ill pregnant with severe COVID-19 ARDS: a case report.

OBJECTIVE: Few data are available on the ICU management and on the possible respiratory complications of invasively ventilated pregnant patients affected by COVID-19 pneumonia, especially in the early phase of pregnancy. Tension pneumothorax has been previously described as a rare cause of respiratory failure after delivery, but its occurrence in the postpartum of COVID-19 patient has not been reported yet. We hereby describe the ICU management of a 23rd gestational week pregnant woman who underwent invasive mechanical ventilation, prone positioning, and cesarean delivery during her ICU stay for COVID-19 related pneumonia. Moreover, we focused on the occurrence and management of recurrent tension pneumothorax after the cesarean delivery. CASE REPORT: A 23rd gestational week pregnant woman was admitted to the ICU for a COVID-19 bilateral pneumonia and underwent invasive mechanical ventilation and prone positioning. Cesarean delivery was planned during the ICU stay, while the patient was receiving invasive mechanical ventilation. After delivery, the patient experienced a recurrent pneumothorax that required the positioning of multiple chest drains. CONCLUSIONS: In pregnant critically ill COVID-19 patients, mechanical ventilation management is particularly challenging, especially in the postpartum period. Prone positioning is feasible and can improve oxygenation and respiratory system compliance, while tension pneumothorax must be suspected if the respiratory function suddenly deteriorates after delivery.

The MAP3K ZAK, a novel modulator of ERK-dependent migration, is upregulated in colorectal cancer.

Often described as a mediator of cell cycle arrest or as a pro-apoptotic factor in stressful conditions, the MAP3K ZAK (Sterile alpha motif and leucine zipper-containing kinase) has also been proven to positively regulate epidermal growth factor receptor (EGFR) and WNT signaling pathways, cancer cell proliferation and cellular neoplastic transformation. Here, we show that both isoforms of ZAK, ZAK-α and ZAK-ß are key factors in cancer cell migration. While ZAK depletion reduced cell motility of HeLa and HCT116 cells, its overexpression triggered the activation of all three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38, as well as an increase in cell motion. On the contrary, the kinase-dead mutants, ZAK-α K45M and ZAK-ß K45M, were not able to provoke such events, and instead exerted a dominant-negative effect on MAPK activation and cell migration. Pharmacological inhibition of ZAK by nilotinib, preventing ZAK-autophosphorylation and thereby auto-activation, led to the same results. Activated by epidermal growth factor (EGF), we further showed that ZAK constitutes an essential element of the EGF/ERK-dependent cell migration pathway. Using public transcriptomic databases and tissue microarrays, we finally established that, as strong factors of the EGFR signaling pathway, ZAK-α and/or ZAK-ß transcripts and protein(s) are frequently upregulated in colorectal adenoma and carcinoma patients. Notably, gene set enrichment analysis disclosed a significant correlation between ZAK+ colorectal premalignant lesions and gene sets belonging to the MAPK/ERK and motility-related signaling pathways of the reactome database, strongly suggesting that ZAK induces such pro-tumoral reaction cascades in human cancers.

Increased severity of dyspeptic symptoms related to mental stress is associated with sympathetic hyperactivity and enhanced endocrine response in patients with postprandial distress syndrome.

BACKGROUND: Mental stress (MS) may alter gastric sensory-motor function. The aim of the study was to assess postprandial autonomic nervous system activity and stress hormones in response to acute mental stress in dyspeptic patients. METHODS: A total of 25 patients with postprandial distress syndrome (PDS; 11 mol L(-1), age 35.9 ± 9.3 years) and 12 healthy controls (5 mol L(-1), age 25.8 ± 4.6 years) underwent electrogastrography and (13) C-octanoate gastric emptying study using a 480 kcal solid meal. Heart rate variability (LF/HF ratio) and corticotrophin-releasing factor, adrenocorticotropic hormone (ACTH), and cortisol serum levels were also evaluated. Dyspeptic symptoms were scored by analogue visual scale and expressed as symptoms total score (TS). The protocol was repeated twice in each subject, with and without a mental stress test before the meal. KEY RESULTS: Mental stress significantly increased postprandial symptoms severity in patients (TS: stress 111 ± 18 vs basal 50 ± 10; P < 0.05). Low-/high-frequency component ratio was significantly higher in patients after MS at 120 min (stress 5.46 ± 0.41 vs basal 3.41 ± 0.64; P < 0.01) and 180 min (stress 5.29 ± 0.2 vs basal 3.58 ± 0.19; P < 0.05). During stress session, in patients we found a significantly higher ACTH level than baseline at 30, 60, 90, 150, 210, 240, and 270 min and a significantly higher cortisol level at 30, 60, 90, 120, 210, and 270 min. Gastric emptying rate and electrical activity were not influenced by MS. CONCLUSIONS & INFERENCES: In PDS patients, administration of MS before meal increases symptoms severity by inducing sympathetic hyperactivity and increased stress hormones levels. As the gastric emptying looks not altered, we conclude that these neurohormonal responses mainly affect sensitive function.

Efficacy of Lactobacillus paracasei sub. paracasei F19 on abdominal symptoms in patients with symptomatic uncomplicated diverticular disease: a pilot study.

AIM: The standard therapeutic approach for symptomatic uncomplicated diverticular disease (DD) remains to be defined, and only a few studies have tested the efficacy of probiotics in these patients. METHODS: Patients with symptomatic uncomplicated DD were randomized to a control arm, i.e., (group A, [N.=16], high-fibre diet alone), or to Group B ([n=18], twice daily 1 sachet of probiotic + high-fibre diet), or group C ([N.=16], twice daily 2 sachets of probiotic + high-fibre diet). The probiotic Genefilus F19© containing Lactobacillus paracasei sub. paracasei F19 was administered for 14 days/month for 6 months. The primary endpoint under consideration was a decrease in abdominal pain and bloating intensity after treatment. RESULTS: Bloating decreased significantly in Groups B and C VAS score group B: 4.6 ± 2.6 vs. 2.3 ± 2.0, P<0.05, group C: 3.9 ± 2.9 vs. 1.8 ± 2.1, P<0.05). The decrease in abdominal pain within 24 hours in these groups did not reach statistical significance. During treatment, none of the group B (N.=4) or group C patients (N=3) with abdominal pain >24 hours reported the recurrence of this symptom, while the 3 group A patients reported at least one episode (P=0.016). No significant difference regarding abdominal pain <24 hours and bloating was observed between the two groups of patients treated with a low or high probiotic dose. CONCLUSION: Lactobacillus paracasei F19, in association with a high-fibre diet, is effective in reducing abdominal bloating and prolonged abdominal pain in symptomatic uncomplicated diverticular disease, and could thus be a promising option in the treatment of these patients.

Quality in emergency departments: a study on 3,285,440 admissions.

INTRODUCTION: A multi-centre study has been conducted, during 2005, by means of a questionnaire posted on the Italian Society of Emergency Medicine (SIMEU) web page. Our intention was to carry out an organisational and functional analysis of Italian Emergency Departments (ED) in order to pick out some macro-indicators of the activities performed. Participation was good, in that 69 ED (3,285,440 admissions to emergency services) responded to the questionnaire. METHODS: The study was based on 18 questions: 3 regarding the personnel of the ED, 2 regarding organisational and functional aspects, 5 on the activity of the ED, 7 on triage and 1 on the assessment of the quality perceived by the users of the ED. RESULTS AND CONCLUSION: The replies revealed that 91.30% of the ED were equipped with data-processing software, which, in 96.83% of cases, tracked the entire itinerary of the patient. About 48,000 patients/year used the ED: 76.72% were discharged and 18.31% were hospitalised. Observation Units were active in 81.16% of the ED examined. Triage programmes were in place in 92.75% of ED: in 75.81% of these, triage was performed throughout the entire itinerary of the patient; in 16.13% it was performed only symptom-based, and in 8.06% only on-call. Of the patients arriving at the ED, 24.19% were assigned a non-urgent triage code, 60.01% a urgent code, 14.30% a emergent code and 1.49% a life-threatening code. Waiting times were: 52.39 min for non-urgent patients, 40.26 min for urgent, 12.08 for emergent, and 1.19 for life-threatening patients.

Intracranial haemorrhage in patients on antithrombotics: clinical presentation and determinants of outcome in a prospective multicentric study in Italian emergency departments.

BACKGROUND: Intracranial haemorrhage (ICH) is the type of stroke associated with the highest death rate, and about 30% of ICH occurs in patients on antithrombotic treatment. This study relates clinical presentations and outcome of ICH patients on oral anticoagulant (OA) or antiplatelet (AP) therapy admitted to 33 Italian emergency departments (ED). METHODS: Consecutive patients were enrolled after cranial computed tomography (CT). Primary outcome was the Modified Rankin Scale (MRS) score at 3 months of follow-up. Common descriptive statistics were computed after stratification for traumatic or spontaneous ICH and identification of the anatomical location of bleeding. Multivariate logistic regression was used to assess predictors of death. RESULTS: We recruited 434 patients on AP therapy and 232 on OA. There were 432 spontaneous and 234 traumatic ICH patients. The proportions of AP and OA patients undergoing neurosurgery were 21.8 and 19.4%, respectively, while < 30% underwent procoagulant medical treatment. At the 3-month follow-up, the case fatality rate was 42.0%, while disability or death (MRS 3-6) was 68.1%. The odds ratio for death in OA versus AP patients was 2.63 (95% CI 1.73-4.00) in the whole population and 2.80 (95% CI 1.77-4.41) in intraparenchymal event patients. Glasgow Coma Scale, age, spontaneous event and anticoagulant use were found to be predictors of death both in traumatic and spontaneous events. CONCLUSION: This study confirms the high prevalence of death or disability in OA and AP patients with ICH. As far as the determinants of mortality and disability are concerned, the results of this study might be useful in the clinical management and allocation of resources in the ED setting. The observed low use of procoagulant therapy highlights the need for ED educational programmes to heighten the awareness of available and effective haemostatic treatments.

Oesophageal acid exposure and altered neurocardiac function in patients with GERD and idiopathic cardiac dysrhythmias.

BACKGROUND: Oesophageal sensory stimuli alter neurocardiac function through autonomic reflexes. AIM: To evaluate in patients with idiopathic supraventricular cardiac dysrhythmias and gastro-oesophageal reflux disease (GERD) whether GE reflux alters neurocardiac function and the effect of acid suppression on cardiac symptoms. METHODS: Thirty-two patients (13 females and 19 males; age: 20-69 years) with dysrhythmias plus GERD, and nine patients (five females and four males; age: 43-58 years) with GERD only, underwent simultaneous 24-h pH-metry and ECG monitoring. Power spectrum analysis of heart rate variability (PSHRV) was obtained with both its low frequency (LF, sympathetic modulation) and high frequency (HF, vagal modulation) components. Hourly mean oesophageal pH and LF/HF ratio were correlated. A 3 months full-dosage PPI therapy (esomeprazole 40 mg/day) was prescribed. RESULTS: In 18 (56%) of the 32 patients with dysrhythmia and in none with GERD only, a significant (P < 0.05) correlation between oesophageal pH and LF/HF ratio (oesophagus-heart correlation) was observed. A significant reduction of cardiac symptoms after PPI therapy was observed only in these patients (13/16 vs. 4/11, P < 0.01). CONCLUSIONS: This study has identified a subgroup of dysrhythmic patients in whom the oesophageal acid stimulus elicited cardiac autonomic reflexes. In these patients acid suppression seems to improve GERD and cardiac symptoms.

Two cases of foodborne botulism with home-preserved asparagus.

Botulism is a rare but potentially fatal disease caused by toxins produced by Clostridium botulinum. We report botulism in two adult females, one of them just tasting from "bad" asparagus and the other eating the full portion. Both patients survived after intermittent mechanical ventilation and trivalent antitoxin administration. The diagnosis was confirmed by detection of botulinum toxin. Acute onset of bilateral cranial neuropathies associated with symmetric descending weakness as well as some key features of the botulism syndrome including absence of fever, symmetric neurologic deficits, the patients remaining responsive and no sensory deficits, with the exception of blurred vision, led to the clinical diagnosis in the first presenting case which was then easily made in the second. Despite the fact that amount of toxin ingested, time-to-symptom development, and time-to-recovery markedly differed in the two patients, their maximal disease severity was similar.

Pathophysiology of gastro-oesophageal reflux disease.

Gastro-oesophageal reflux disease is a condition in which the reflux of gastric contents into the oesophagus provokes symptoms or complications and impairs quality of life. Typical symptoms of gastro-oesophageal reflux disease are heartburn and regurgitation but gastro-oesophageal reflux disease has also been related to extra-oesophageal manifestations, such as asthma, chronic cough and laryngitis. The pathogenesis of gastro-oesophageal reflux disease is multifactorial, involving transient lower oesophageal sphincter relaxations and other lower oesophageal sphincter pressure abnormalities. As a result, reflux of acid, bile, pepsin and pancreatic enzymes occurs, leading to oesophageal mucosal injury. Other factors contributing to the pathophysiology of gastro-oesophageal reflux disease include hiatal hernia, impaired oesophageal clearance, delayed gastric emptying and impaired mucosal defensive factors. Hiatal hernia contributes to gastro-oesophageal reflux disease by promoting lower oesophageal sphincter dysfunction. Impaired oesophageal clearance is responsible for prolonged acid exposure of the mucosa. Delayed gastric emptying, resulting in gastric distension, can significantly increase the rate of transient lower oesophageal sphincter relaxations, contributing to postprandial gastro-oesophageal reflux disease. The mucosal defensive factors play an important role against development of gastro-oesophageal reflux disease, by neutralizing the backdiffusion of hydrogen ion into the oesophageal tissue. While the pathogenesis of oesophageal symptoms is now well known, the mechanisms underlying extra-oesophageal airway manifestations are still poorly understood. Two hypotheses have been proposed: direct contact of gastric acid with the upper airway and a vago-vagal reflex elicited by acidification of the distal oesophagus, leading to bronchospasm. In conclusion, gastro-oesophageal reflux disease can be considered as the result of a complex interplay of factors, all promoting the contact of gastric acidic contents with the oesophageal mucosa, leading to different degrees of oesophageal damage.

Medical treatment of gastro-oesophageal reflux disease.

The introduction, in the last two decades, of strongly effective acid suppressant drugs, such as proton pump inhibitors has radically modified the way of treating gastro-oesophageal reflux disease. In clinical trials, these agents have constantly been demonostrated to be more effective than other acid-suppressant agents such as H2-receptor antagonists in relief of symptoms and healing of oesophagitis, the two main goals of gastro-oesophageal reflux disease treatment. They provide a prompt clinical benefit to most patients and can be safely used in long-term gastro-oesophageal reflux disease management for maintenance of clinical and endoscopic remission, because of their negligible adverse-events profile. Therapeutic protocols vary depending on the severity of symptoms and the degree of oesophageal injury. In patients with mild symptoms and with minimal lesions at endoscopy, a "step-down" therapy, in the short-term, is considered the best medical strategy, while in the long-term the therapy "on-demand" appears to be a reasonable approach. Patients with non-erosive disease seem to have a lower response rate to proton pump inhibitor treatment. More severe grades of oesophagitis must be treated with full-dose proton pump inhibitors without withdrawal. Data on the treatment of extra-oesophageal manifestations of gastro-oesophageal reflux disease are few and controversial. Overall, it appears that patients with extra-oesophageal symptoms of gastro-oesophageal reflux disease must be treated with higher doses of pharmacological treatment, principally with proton pump inhibitors, and with longer periods of treatment to achieve complete relief of symptoms, as compared with patients with typical symptoms of gastro-oesophageal reflux disease and erosive oesophagitis.

Validity of Lot Quality Assurance Sampling to optimize falciparum malaria surveys in low-transmission areas.

To control the reappearance of malaria in the Madagascan highlands, indoor house-spraying of DDT was conducted from 1993 until 1998. Before the end of the insecticide-spraying programme, a surveillance system was set up to allow rapid identification of new malaria epidemics. When the number of suspected clinical malaria cases notified to the surveillance system exceeds a predetermined threshold, a parasitological survey is carried out in the community to confirm whether or not transmission of falciparum malaria is increasing. Owing to the low specificity of the surveillance system, this confirmation stage is essential to guide the activities of the control programme. For this purpose, Lot Quality Assurance Sampling (LQAS), which usually requires smaller sample sizes, seemed to be a valuable alternative to conventional survey methods. In parallel to a conventional study of Plasmodium falciparum prevalence carried out in 1998, we investigated the ability of LQAS to rapidly classify zones according to a predetermined prevalence level. Two prevalence thresholds (5% and 15%) were tested using various sampling plans. A plan (36, 2), meaning that at least 2 individuals found to be positive among a random sample of 36, enabled us to classify a community correctly with a sensitivity of 100% and a specificity of 94%. LQAS is an effective tool for rapid assessment of falciparum malaria prevalence when monitoring malaria transmission.

Malaria in the highlands of Madagascar after five years of indoor house spraying of DDT.

The central region of Madagascar is a vast area of highlands (altitude 700-2000 m). Malaria transmission has re-established itself here since the last epidemic of 1985-90 and has caused the deaths of 40,000 persons according to the Minister of Health. To combat the main malaria vector in the region, Anopheles funestus, annual programmes of indoor house spraying of DDT were carried out between December 1993 and January 1998 in most rural areas at altitude 1000-1500 m. A parasitological and serological study was then conducted in the highland schools to evaluate the impact of the programme and set up a database on the region. Using a cluster-sampling method 2 independent selections were conducted (one of 130 sites, the other of 40 sites). During the study, 13,462 schoolchildren were examined, 71% living in sprayed villages. Parasite prevalence among schoolchildren declined as altitude increases, from 11% at 700-900 m to 0.4% at > 1500 m. Below 1500 m, the impact of the spraying on the prevalence of the parasite was very clear (an average decrease of from 20% to 2.7% below 1000 m and of from 4.5% without spraying to 0.8% at 1000-1500 m). Geographical analysis of the data showed that the marginal regions remained the most affected by malaria (especially outside spraying zones), and persistence of 'pockets of transmission' at 1000-1500 m, essentially in areas where spraying has never been used. In 9 schools, anti-Plasmodium antibodies were sought by indirect immunofluorescence on thick smears of parasitized red blood cells. The seroprevalence ranged from 22% to 63%, which suggests that the parasite is still circulating in the region. Even though our data show that vector control continues to be very successful in the Madagascan highlands, rapid reinfection could occur and must be monitored following spraying. To this end, the Minister for Health, with the support of the Italian Co-operation, has placed the region under epidemiological surveillance since 1997. An alert system for the timely detection of the sources of epidemics and the targeting of the antivectoral campaign is also in operation. Our study suggests that this strategy should be reinforced by the spraying of DDT in the marginal zones in order to consolidate the results obtained at higher altitudes.

[Epidemiologic surveillance system and control of malaria in the central highlands of Madagascar: results 1999-2000]. / Système de surveillance épidémiologique et d'alerte du paludisme sur les Hautes Terres Centrales de Madagascar: résultats 1999-2000.

The central highlands in Madagascar are characterized by an unstable occurrence of malaria with the risk of sporadic outbreaks. In major parts of the region DDT indoor spraying campaigns have been carried out from 1993 to 1998. This strategy was in 1999 replaced by another anti-vector intervention program targeting residual foci as detected by a surveillance and early warning system. This system is based on monitoring of presumptive malaria cases in the communities by which the number of presumptive cases exceeded a defined warning threshold value per month. The system was in the follow-up period shown to be very sensitive to variation of the coverage of anti-vector interventions: the number of presumptive cases decreased in the villages in which indoor spraying had been carried out and a minor increase was observed in those villages, where indoor spraying has been suspended. An increase of malaria cases was observed in 44 (20.8%) out of 212 study sites in the same period. The increase was in particular predominant in areas at lower attitude at the outer zones of the central highlands.

Electrical coupling and plasticity of the mitochondrial network.

Kinetic fluorescence imaging and the potentiometric probe tetramethylrhodamine methyl ester (TMRM) were used to evoke and detect changes in membrane potential (delta Psi(m)) of individual mitochondria in living cells. As a combined effect of preferential TMRM accumulation in mitochondria, and of TMRM photoactivation, individual organelles displayed sharp transient depolarizations caused by local reactive oxygen species (ROS)-mediated gatings of the mitochondrial permeability transition pore (PTP). In COS-7 cells, such directed repetitive gatings of the PTP gave rise to stochastic delta Psi(m)flickering at the level of individual organelles, but also to prominent synchronous delta Psi(m)transitions in whole subgroups of the mitochondrial population, indicative of the existence of an underlying electrically coupled mitochondrial network. In single cells, this network could comprise as much as 65% of the total mitochondrial population, a nd exhibited a high plasticity with mitochondrial units spontaneously connecting to and disconnecting from the coupled structure within seconds. These results indicate that in resting cells, the mitochondrial network is a dynamic proton-conducting structure capable to commute and coordinate electrical signals generated by the PTP.

A genetically encoded, fluorescent indicator for cyclic AMP in living cells.

Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.

Control of epidemic malaria on the highlands of Madagascar.

The Malagashy national malaria control programme ('Programme National de Lutte contre le Paludisme', PNLP) has been developing, since 1996, an epidemiological early warning system for malaria epidemics in the Central Highlands with the support of the Italian Development Cooperation. The system is based on the monitoring of malaria morbidity (clinical diagnosis) in 536 peripheral health centres (CSB) of the Highlands. The intervention area corresponds to 27 districts of the Antananarivo and Fianarantsoa provinces (4.7 million inhabitants) and spans around 100,000 square km. For each CSB a monthly warning threshold, defined as the 1993-1996 monthly mean number of malaria cases plus two standard deviations, was established. Three levels of epidemic alert have been defined according to the number of times the cases of presumptive malaria surpassed the threshold and according to the reported presence of severe malaria cases. The surveillance system relies also on the monitoring, in district hospitals of the Highlands, of the Plasmodium falciparum infection rate among clinically diagnosed malaria cases. A total of 185,589 presumptive malaria cases, corresponding to a 42/1000 malaria incidence, were recorded in 1997 by the surveillance system. During the same year 184 alerts of 2nd degree were reported. During 1998 173,632 presumptive malaria cases corresponding to a 38/1000 incidence were reported and 207 alerts of 2nd degree were detected; 75 of these alerts were investigated with ad hoc surveys and 3 initial malaria epidemics identified and controlled. Out of 6884 presumptive malaria cases diagnosed in the district hospitals during 1997-1998, only 835 (12.1%) have been confirmed by microscopy (P. falciparum 81.7%, P. vivax 15.0%, P. malariae 2.5%, P. ovale 0.2%, mixed infections 0.6%); 22.4% of these infections were imported cases from coastal endemic areas. The efficiency of the system in monitoring the trend of malaria morbidity and in the rapid detection and response to malaria epidemics is still being evaluated.

Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes.

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.