Keratoconus and Personality Traits: A Case-Control Study.

PURPOSE: The aim of this study was to delineate the personality traits of patients affected by keratoconus (KC) compared with a group of nonkeratoconic controls matched in age and sex. METHODS: In this prospective interventional case-control study, 60 consecutive subjects (30 KC cases and 30 healthy controls), aged 18 to 30, were enrolled at the time of their first encounter at the ophthalmology unit of the Fondazione Policlinico "Tor Vergata", Roma. After completing the ophthalmic evaluation, participants were asked to respond to the National Eye Institute Visual Function Questionnaire-25 (NEI VFQ-25). A complete psychiatric assessment was performed, including the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (SCID-5); the Symptom Check List-90-Revised (SCL-90); the Temperament Evaluation of Memphis, Pisa, Paris, and San Diego-Modified (TEMPS-M); and the NEO Five-Factor Inventory (NEO-FFI). RESULTS: Cases had lower quality of life than controls, as demonstrated by lower scores in all NEI VFQ-25 subdomains. Nine patients with KC (30.0%) were diagnosed by the SCID-5 with at least 1 cluster C personality disorder, resulting in a 9-fold increased risk compared with controls. Moreover, keratoconic patients showed a more pronounced psychosomatic symptomatology (SCL-90) and a characteristic neurotic temperament (TEMPS-M and NEO-FFI). CONCLUSIONS: Our results support the hypothesis that subjects with KC feature dysfunctional coping mechanisms and personality traits, which might already be present at the first clinical encounter. Ophthalmologists should question the mental and emotional status of patients with KC and be especially careful in managing these patients.

EBEI: A new index of eye bank efficiency.

PURPOSE: To describe a new proxy of the Eye Bank (EB) activity named "Eye Bank Efficiency Index" (EBEI), calculated as the ratio between the number of corneal tissues distributed by an EB within a certain time period, and the difference between the number of procured and discarded tissues. METHODS: To demonstrate the effectiveness of the new metric as compared to traditional statistics, an analysis was conducted using data from the largest Italian Eye Bank (Fondazione Banca degli Occhi del Veneto Venice, Italy). Collected data included: the number of corneas retrieved, the number of discarded grafts, and the number of distributed tissues. The analysis focused on three defined timeframes: January to December, March to May (the "Italian Lockdown period"), and June to December (the "Italian post-lockdown period"). RESULTS: In 2020, the annual variation of the EBEI showed a significant increase of up to 3.4% compared to the previous year (0.986 in 2019; 1.020 in 2020), but then gradually decreased to 0.993 in 2022. However, during the months of lockdown in 2020, there was a significant decline of -13.8% in the EBEI compared to the same period in the previous year. The variation in the EBEI during the post-lockdown months was minimal in 2020 and 2021, with the lowest EBEI value of 0.976 being reached in 2022 (-7.8% compared to 2019). CONCLUSION: The EBEI is a simple and reliable new measure of the EB activity. Its widespread adoption could ensure a more accurate and reliable analysis of EB data for academic, political, and economic purposes.

Lax eyelid condition (LEC) and floppy eyelid syndrome (FES) prevalence in obstructive sleep apnea syndrome (OSA) patients: a systematic review and meta-analysis.

PURPOSE: Lax eyelid condition (LEC) and floppy eyelid syndrome (FES) represent two distinct conditions which have been associated with several ocular and systemic comorbidities. The main aim of this systematic review and meta-analysis is to explore the available literature to estimate the prevalence rate of LEC and FES in obstructive sleep apnea (OSA). METHODS: The protocol of this systematic review and meta-analysis has been registered in PROSPERO. Four electronic databases (PubMed/MEDLINE, Google Scholar, Cochrane Library, Web of Science) were searched from inception to December 24, 2021. A random intercept logistic regression model was carried out for the analysis of overall proportions. Odds ratio and mean difference were reported as measures of the effect size in the presence of binary and continuous outcomes, respectively. The estimated numbers of LEC/FES patients in OSA were calculated by multiplying the prevalence rate determined by our random-effects model and the corresponding Benjafield et al.'s population prospect. RESULTS: We included 11 studies comprising 1225 OSA patients of whom 431 and 153 affected by LEC and FES, respectively. Our model estimated a pooled prevalence rate for LEC and FES in OSA patients of 40.2% (95%CI: 28.6-53.1%) and of 22.4% (95%CI: 13.8-34.2%), respectively. The number of LEC/FES affected individuals among OSA patients is expected to peak up to 376 and to 210 million, respectively. OSA patients appeared to have a 3.4 (95%CI: 2.2-5.2) and a 3.0 (95%CI: 1.7-5.5) increased risk of developing LEC and FES than the healthy counterpart. CONCLUSION: Prevalence of LEC and FES is higher in OSA-affected patients compared to controls. More studies are warranted to investigate the mechanisms leading to the development of LEC and/or FES in OSA patients, as well as the feasibility of the adoption of these clinical findings as screening tools for OSA.

Global Prevalence of Fuchs Endothelial Corneal Dystrophy (FECD) in Adult Population: A Systematic Review and Meta-Analysis.

Purpose: To evaluate the global prevalence of Fuchs endothelial corneal dystrophy (FECD). Design: Systematic review and meta-analysis. Methods: A systematic electronic literature search was conducted on PubMed/MedLine, Cochrane Library, and Google Scholar, in order to select papers analysing the prevalence rate of FECD. Two authors independently conducted the electronic search. After removal of duplicates, title and abstract screening, and full-text analysis, data from selected articles were archived in a customized Excel spreadsheet. Risk of bias assessment was performed using the Joanna Briggs Institute Prevalence Critical Appraisal Tool. Meta-analysis was conducted using R (version 1.4.1106, package "meta"). Results: A total of 6660 eligible articles were retrieved from the initial electronic search. Only 4 original works were included in the qualitative and quantitative analysis. Of the 4746 patients included in this meta-analysis (i.e., 2232 male (M) and 2322 female (F)), we retrieved 269 FECD cases (81 M; 188 F), with a pooled prevalence estimates of 7.33% (95% CI: 4.08-12.8%). Statistically significant gender-related differences in the prevalence of FECD emerged by the analysis (OR: 2.22; 95% CI: 1.66-2.96, p=0.0016). While the total number of people aged >30 years with FECD is nowadays estimated at nearly 300 million, an increase of 41.7% in the number of FECD-affected patients is expected by 2050, when the overall figure is supposed to rise up to 415 million. Conclusion: This study provides a reliable figure of the present and future epidemiological burden of FECD globally, which might be useful for the design of FECD screening, treatment, rehabilitation, and related public health strategies.

Molecular validation of pathogen-reduction technologies using rolling-circle amplification coupled with real-time PCR for torquetenovirus DNA quantification.

BACKGROUND: Pathogen reduction technologies (PRT) based on nucleic-acid damaging chemicals and/or irradiation are increasingly being used to increase safety of blood components against emerging pathogens, such as convalescent plasma in the ongoing COVID-19 pandemic. Current methods for PRT validation are limited by the resources available to the blood component manufacturer, and quality control rely over pathogen spiking and hence invariably require sacrifice of the tested blood units: quantitative real-time PCR is the current pathogen detection method but, due to the high likelihood of detecting nonviable fragments, requires downstream pathogen culture. We propose here a new molecular validation of PRT based on the highly prevalent human symbiont torquetenovirus (TTV) and rolling circle amplification (RCA). MATERIALS AND METHODS: Serial apheresis plasma donations were tested for TTV before and after inactivation with Intercept® PRT using real-time quantitative PCR (conventional validation), RCA followed by real-time PCR (our validation), and reverse PCR (for cross-validation). RESULTS: While only 20% of inactivated units showed significant decrease in TTV viral load using real-time qPCR, all donations tested with RCA followed by real-time PCR showed TTV reductions. As further validation, 2 units were additionally tested with reverse PCR, which confirmed absence of entire circular genomes. DISCUSSION: We have described and validated a conservative and easy-to-setup protocol for molecular validation of PRT based on RCA and real-time PCR for TTV.

Visuomotor Interactions and Perceptual Judgments in Virtual Reality Simulating Different Levels of Gravity.

Virtual reality is used to manipulate sensorimotor interactions in a controlled manner. A critical issue is represented by the extent to which virtual scenarios must conform to physical realism to allow ecological human-machine interactions. Among the physical constraints, Earth gravity is one of the most pervasive and significant for sensorimotor coordination. However, it is still unclear whether visual perception is sensitive to the level of gravity acting on target motion displayed in virtual reality, given the poor visual discrimination of accelerations. To test gravity sensitivity, we asked participants to hit a virtual ball rolling down an incline and falling in air, and to report whether ball motion was perceived as natural or unnatural. We manipulated the gravity level independently for the motion on the incline and for the motion in air. The ball was always visible during rolling, whereas it was visible or occluded during falling before interception. The scene included several cues allowing metric calibration of visual space and motion. We found that the perception rate of natural motion was significantly higher and less variable when ball kinematics was congruent with Earth gravity during both rolling and falling. Moreover, the timing of target interception was accurate only in this condition. Neither naturalness perception nor interception timing depended significantly on whether the target was visible during free-fall. Even when occluded, free-fall under natural gravity was correctly extrapolated from the preceding, visible phase of rolling motion. Naturalness perception depended on motor performance, in addition to the gravity level. In sum, both motor and perceptual responses were guided by an internal model of Earth gravity effects. We suggest that, in order to enhance perceptual sensitivity to physical realism, virtual reality should involve visual backgrounds with metric cues and closed-loop sensorimotor interactions. This suggestion might be especially relevant for the design of rehabilitation protocols.

Rolling Motion Along an Incline: Visual Sensitivity to the Relation Between Acceleration and Slope.

People easily intercept a ball rolling down an incline, despite its acceleration varies with the slope in a complex manner. Apparently, however, they are poor at detecting anomalies when asked to judge artificial animations of descending motion. Since the perceptual deficiencies have been reported in studies involving a limited visual context, here we tested the hypothesis that judgments of naturalness of rolling motion are consistent with physics when the visual scene incorporates sufficient cues about environmental reference and metric scale, roughly comparable to those present when intercepting a ball. Participants viewed a sphere rolling down an incline located in the median sagittal plane, presented in 3D wide-field virtual reality. In different experiments, either the slope of the plane or the sphere acceleration were changed in arbitrary combinations, resulting in a kinematics that was either consistent or inconsistent with physics. In Experiment 1 (slope adjustment), participants were asked to modify the slope angle until the resulting motion looked natural for a given ball acceleration. In Experiment 2 (acceleration adjustment), instead, they were asked to modify the acceleration until the motion on a given slope looked natural. No feedback about performance was provided. For both experiments, we found that participants were rather accurate at finding the match between slope angle and ball acceleration congruent with physics, but there was a systematic effect of the initial conditions: accuracy was higher when the participants started the exploration from the combination of slope and acceleration corresponding to the congruent conditions than when they started far away from the congruent conditions. In Experiment 3, participants modified the slope angle based on an adaptive staircase, but the target never coincided with the starting condition. Here we found a generally accurate performance, irrespective of the target slope. We suggest that, provided the visual scene includes sufficient cues about environmental reference and metric scale, joint processing of slope and acceleration may facilitate the detection of natural motion. Perception of rolling motion may rely on the kind of approximate, probabilistic simulations of Newtonian mechanics that have previously been called into play to explain complex inferences in rich visual scenes.

Intercepting virtual balls approaching under different gravity conditions: evidence for spatial prediction.

To accurately time motor responses when intercepting falling balls we rely on an internal model of gravity. However, whether and how such a model is also used to estimate the spatial location of interception is still an open question. Here we addressed this issue by asking 25 participants to intercept balls projected from a fixed location 6 m in front of them and approaching along trajectories with different arrival locations, flight durations, and gravity accelerations (0g and 1g). The trajectories were displayed in an immersive virtual reality system with a wide field of view. Participants intercepted approaching balls with a racket, and they were free to choose the time and place of interception. We found that participants often achieved a better performance with 1g than 0g balls. Moreover, the interception points were distributed along the direction of a 1g path for both 1g and 0g balls. In the latter case, interceptions tended to cluster on the upper half of the racket, indicating that participants aimed at a lower position than the actual 0g path. These results suggest that an internal model of gravity was probably used in predicting the interception locations. However, we found that the difference in performance between 1g and 0g balls was modulated by flight duration, the difference being larger for faster balls. In addition, the number of peaks in the hand speed profiles increased with flight duration, suggesting that visual information was used to adjust the motor response, correcting the prediction to some extent.NEW & NOTEWORTHY Here we show that an internal model of gravity plays a key role in predicting where to intercept a fast-moving target. Participants also assumed an accelerated motion when intercepting balls approaching in a virtual environment at constant velocity. We also show that the role of visual information in guiding interceptive movement increases when more time is available.

Pre-birth selection of umbilical cord blood donors.

BACKGROUND: . The fact that only a small percentage of cord blood units (CBU) stored are actually used for transplantation contributes to raising the already high costs of their processing and cryopreservation. The identification of predictors allowing the early identification of suitable CBU would allow a reduction of costs for the collection, storage and characterisation of CBU with insufficient volume or cell numbers. In our bank we have adopted a cut-off value for using CBU of 8 x 10(8) nucleated cells and a volume >or= 60 mL. MATERIALS AND METHODS: In 365 banked CBU, we evaluated the correlation between neonatal/gestational parameters and laboratory data used to assess their quality. RESULTS: Biparietal diameter (BPD) and abdominal circumference were significantly and positively correlated with CBU volume (r(2)=0.12, p=0.0011 and r(2)=0.092, p=0.0063, respectively). Receiver operating characteristic (ROC) analysis showed that both parameters can be used to identify CBU with insufficient volume (BPD: area under the curve 0.69, 95% CI=0.57-0.82, p=0.004; abdominal circumference: area under the curve 0.67, 95% CI=0.54-0.79, p<0.01). BPD and head circumference, but not abdominal circumference or femoral length, were positively correlated with white blood cell (WBC) count (r(2)=0.215, p=0.031, and r(2)=0.299, p=0.015, respectively). Abdominal circumference, but not BPD, head circumference or femoral length, was statistically significantly correlated with the number of CD34(+) cells in the CBU. Weight at birth and placental weight were positively correlated with WBC count, blood volume, CD34(+) cell count, total colony-forming units and burst-forming units. CONCLUSION: . Pre-birth assessment of BPD might allow the selection of donors who would yield CBU of sufficient volume and WBC count and avoid the costs of collecting, transferring, storing and analysing CBU with a high probability of resulting unsuitable for transplantation.

Urokinase expression in course of benign and malignant mammary lesions: comparison between nodular and healthy tissues.

PURPOSE: Increased expression of urokinase (uPA), a member of the serine protease family, is an effector of metastatic cascade and has been reported in various malignancies, including breast cancer. uPA overexpression in cancer tissues was correlated with a more aggressive phenotype and it is considered a strong and independent unfavorable prognostic factor in breast cancer. METHODS: Using real-time PCR assay, we analyzed uPA expression of malignant and benign breast nodular lesions versus healthy tissues (normal breast and lymphocytes). RESULTS: We found that besides breast cancer nodule, normal mammary gland and lymphocytes overexpressed uPA too. Tissues obtained from women with benign lesions expressed homogeneous and lower uPA. CONCLUSIONS: In conclusion, although uPA overexpression is typical of cancer tissues, it could be considered as a feature of the whole organism affected by cancer. On the basis of these first results, uPA could be considered for further studies as a possible useful therapeutic target in breast cancer.

Colorectal cancer after breast cancer: magnitude of risk in clinical practice and in the literature.

AIMS AND BACKGROUND: The aim of our study was to estimate the incidence of second primary tumors among breast cancer patients from the Division of Oncology of Andosilla Hospital (Viterbo, Italy). In particular, we studied the relationship between breast and colorectal cancer. METHODS AND STUDY DESIGN: Eligible women were those with primary invasive breast cancer who had been treated and/or followed up at our division. We compared our data with those reported in the literature. RESULTS: Of 114 women with breast cancer, 21 (18.5%) developed multiple primary cancers, with colorectal cancer accounting for a quarter. We found a higher incidence of colorectal cancer than reported in the literature (5% vs 0.66-1%). At the moment we are not able to explain this difference. CONCLUSIONS: Quite a few studies reported a link between breast and colorectal cancer, but the magnitude of the risk (standardized incidence ratio 1.3-2) does not justify a screening program for colorectal disease in breast cancer patients. However, considering that the risk is small but not negligible--along with the high incidence of colorectal adenomas described in breast cancer patients and the possible existence of common risk factors--we invite clinicians not to neglect the possibility of a colorectal cancer diagnosis in women who have had breast cancer.

[Diagnosis and treatment of fibroadenoma of the breast: 20 years' experience]. / Diagnosi e trattamento del fibroadenoma della mammella: 20 anni di esperienza.

The diagnostic differentiation of breast lesions is very important because of the frequency with which they occur. Though fibroadenoma is easy to diagnose, some cases prove really hard to distinguish. Therefore, various methods have been suggested both for diagnosis and therapy, but no common approach has been achieved to date. We report our experience with 1350 cases diagnosed over a twenty-year period. The diagnosis was made on the basis of a diagnostic protocol drawn up over the years where cytology is of primary importance. With regard to therapy, we decide to operate when the cytological findings prompt the need for a histological examination of the lesion. Moreover, we operate on those cases where either an increase in size or a morphological change of the lesion has taken place. Histological examinations were carried out in 420 cases out of 1350 and only one of these cases turned out to be a carcinoma. As a result, the 0.24% error in the diagnosis can be considered irrelevant. In spite of the different therapies suggested (cryoablation, laser hyperthermia, i.a.), we believe that surgical treatment with local anaesthetic is the most suitable solution in those cases requiring treatment. Obviously, the diagnosis has to be accurate and made by surgeons with appropriate expertise. When the diagnosis is certain and the conditions of the lesion are stable, the best policy seems to be periodic follow-up.

Immunohistochemical localization of 3-nitrotyrosine in the nasal respiratory mucosa of patients with vasomotor rhinitis.

CONCLUSION: This study demonstrates that, in the nasal respiratory mucosa of patients with vasomotor rhinitis, oxidative stress following peroxynitrite formation is confined to the respiratory epithelium. This suggests that the role of peroxynitrite in vasomotor rhinitis differs from its role in other diseases of the respiratory tract. The results of this study also support the concept that different pathogenetic mechanisms are probably involved in vasomotor rhinitis. OBJECTIVE: Previous studies indicated that nitric oxide (NO) is involved in the pathogenesis of vasomotor rhinitis, strong expression of NO synthase being detected in the smooth muscle cells of the cavernous sinuses and in the respiratory epithelium. However, most adverse effects of high levels of NO originate from the reaction of NO with superoxide anions to form peroxynitrite. Therefore, in this study we evaluated the involvement of peroxynitrite in the pathogenesis of vasomotor rhinitis. MATERIAL AND METHODS: Sites of peroxynitrite formation were identified by immunolabelling for 3-nitrotyrosine (3NT), its footprint in tissues. Samples of nasal mucosa were obtained from vasomotor rhinitis patients and from control subjects who had undergone corrective surgery of the nasal septum. All samples were obtained by reduction of the inferior turbinate. RESULTS: Examination of specimens from vasomotor rhinitis patients revealed that 3NT is absent in epithelium with a normal appearance, cells of the subepithelial connective tissue, the glands and the blood vessels, including the cavernous sinuses. In contrast, intense 3NT immunolabelling was found in the disrupted respiratory epithelium. 3NT was not present in any of the specimens from control subjects.

One-day surgery in a series of 150 breast cancer patients: efficacy and cost-benefit analysis.

Financing health-care expenditure has become increasingly more difficult. Considering the high frequency of breast cancer, which affects one million women in the world each year, the reductions of medical expenditure for the treatment of this disease is highly desirable within the limits of medical efficiency and safety. One hundred and fifty patients with carcinoma of the breast underwent surgery in our department with one-day hospitalization. Patients were discharged with the drainage tube still in place and later treated in the out-patient setting, for dressing and checking the wound, and removing the stitches and drainage tube. Four cases of seroma were registered, all resolved by aspiration of the fluid in a single visit, 1 case of haematoma and 1 case of infection. Patients who underwent this short-stay treatment were amply satisfied. Our experience demonstrates that this type of treatment is both safe and effective. Moreover, it provides considerable benefits in terms of national health-care costs as well as being psychologically better for the patients.

Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation.

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.

Alkylation of sulfhydryl groups on Galpha(s/olf) subunits by N-ethylmaleimide: regulation by guanine nucleotides.

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells.

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.