Recently we demonstrated that PLC⑀ plays an important role in ␤-adrenergic receptor (␤AR) stimulation of Ca 2؉-induced Ca 2؉ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of ␤AR involving the cAMPdependent Rap GTP exchange factor, Epac, and PLC⑀ regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epacselective cAMP analog, 8-4-(chlorophenylthio)-2-O-methyladenosine-3,5-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca 2؉ transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC⑀ ؊/؊ cardiac myocytes but rescued by transduction with PLC⑀, indicating that Epac is upstream of PLC⑀. Furthermore, transduction of PLC⑀ ؉/؉ cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that ␤AR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLC⑀ pathway. We also show that Epac/PLC⑀-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca 2؉ clearance mechanisms. These data define a novel endogenous PKA-independent ␤AR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC⑀ that enhances intracellular Ca 2؉ release in cardiac myocytes.Stimulation of adrenergic receptors by either neurohumoral or systemic release of the catecholamines epinephrine and norepinephrine produces acute increases in cardiac contractility during stress and exercise to increase cardiac output and oxygen delivery to tissues. Much of the increase in cardiac output is due to the direct stimulation of the ␤ adrenergic receptor (␤AR) 2 in cardiac myocytes (1, 2). Activation of ␤AR activates G s and adenylyl cyclase resulting in the production of cAMP and subsequent activation of protein kinase A, which phosphorylates key components of the calcium handling and contractile machinery.Analysis of a phospholipase C (PLC)⑀ knock-out mouse model (PLC⑀ Ϫ/Ϫ ) generated in our laboratory indicates that PLC⑀ contributes to ␤AR-dependent regulation of cardiac function (3). PLC⑀ Ϫ/Ϫ mice exhibit significantly decreased left ventricular developed pressure in response to acute stimulation with the ␤AR agonist, isoproterenol. Isolated myocytes from PLC⑀ Ϫ/Ϫ mice exhibit decreased isoproterenol-dependent enhancement of electrically evoked Ca 2ϩ release in the absence of effects on ␤AR density or cAMP generation. These data implicate PLC⑀ as a novel component of ␤AR regulation of Ca 2ϩ release, which had not previously been described in the heart. However, the pathway linking ␤AR stimulation to PLC⑀ activation is unknown.PLC-mediated phosphatidyl-4,5-bisphosphate hydrolysis resulting in intracellular Ca 2ϩ release and protein kinase C activation is an integral signaling component of many physiological processes in a variety of tissu...

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Phospholipase C⑀ (PLC⑀) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLC␤ isoforms through G q family G proteins and G␤␥ subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLC⑀ and the PLC␤ isoform, PLC␤3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist-and isoform-specific profiles. PLC␤3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLC⑀, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLC⑀ in knockdown cells dosedependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLC⑀ and PLC␤3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLC⑀ or PLC␤3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLC⑀ and PLC␤3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLC␤3 is predominantly involved in acute and PLC⑀ in sustained PI hydrolysis.The phosphatidylinositol-specific phospholipase C (PLC) 2 family is a group of critical cellular signaling enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate (PI) to generate inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol, which increase the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) and activate protein kinase C, respectively (1, 2). Eleven isoforms of PLC, representing five distinct, differentially regulated classes, have been identified: PLC␤1 to ␤4; PLC␥1 and ␥2; PLC␦1, ␦3, and ␦4; and PLC⑀ and PLC. PLC␤ is regulated by G-protein-coupled receptor (GPCR) activation of heterotrimeric G q family G-proteins and G␤␥ subunits. PLC␥ is regulated by tyrosine phosphorylation by receptor tyrosine kinases (e.g. epidermal growth factor and plateletderived growth factor) and nonreceptor tyrosine kinases (e.g. Src) activated by immunoglobulin and cytokines. Regulation of PLC␦ is less well understood but is probably regulated by changes in [Ca 2ϩ ] i , possibly downstream from activation of other PLC isoforms, and by high molecular weight G-protein, G h . PLC is also regulated by [Ca 2ϩ ] i (3). PLC⑀ was discovered only recently and is the largest member of the PLC family (4 -7). PLC⑀ is regulated by the monomeric Ras (4, 8, 9) and Rho (9, 10) families, the heterotrimeric G12 family (5, 9), and G␤␥ sub...

Objective: Pneumonia is the leading cause of pediatric mortality worldwide among children 0–5 years old. Lung ultrasound can be used to diagnose pneumonia in rural areas as it is a portable and relatively economic imaging modality with ~95% sensitivity and specificity for pneumonia in children. Lack of trained sonographers is the current limiting factor to its deployment in rural areas. In this study, we piloted training of a volume sweep imaging (VSI) ultrasound protocol for pneumonia detection in Peru with rural health workers. VSI may be taught to individuals with limited medical/ultrasound experience as it requires minimal anatomical knowledge and technical skill. In VSI, the target organ is imaged with a series of sweeps and arcs of the ultrasound probe in relation to external body landmarks. Methods: Rural health workers in Peru were trained on a VSI ultrasound protocol for pneumonia detection. Subjects were given a brief didactic session followed by hands-on practice with the protocol. Each attempt was timed and mistakes were recorded. Participants performed the protocol until they demonstrated two mistake-free attempts. Results: It took participants a median number of three attempts (range 1–6) to perform the VSI protocol correctly. Time to mastery took 51.4 ± 17.7 min. There were no significant differences among doctors, nurses, and technicians in total training time (P = 0.43) or number of attempts to success (P = 0.72). Trainee age was not found to be significantly correlated with training time (P = 0.50) or number of attempts to success (P = 0.40). Conclusion: Rural health workers learned a VSI protocol for pneumonia detection with relative ease in a short amount of time. Future studies should investigate the clinical efficacy of this VSI protocol for pneumonia detection. Key Message: A volume sweep imaging (VSI) protocol for pneumonia detection can be taught with minimal difficulty to rural health workers without prior ultrasound experience. No difference was found in training performance related to education level or age. VSI involves no significant knowledge of anatomy or technical skill.

BackgroundRespiratory illness is a leading cause of morbidity in adults and the number one cause of mortality in children, yet billions of people lack access to medical imaging to assist in its diagnosis. Although ultrasound is highly sensitive and specific for respiratory illness such as pneumonia, its deployment is limited by a lack of sonographers. As a solution, we tested a standardised lung ultrasound volume sweep imaging (VSI) protocol based solely on external body landmarks performed by individuals without prior ultrasound experience after brief training. Each step in the VSI protocol is saved as a video clip for later interpretation by a specialist.MethodsDyspneic hospitalised patients were scanned by ultrasound naive operators after 2 hours of training using the lung ultrasound VSI protocol. Separate blinded readers interpreted both lung ultrasound VSI examinations and standard of care chest radiographs to ascertain the diagnostic value of lung VSI considering chest X-ray as the reference standard. Comparison to clinical diagnosis as documented in the medical record and CT (when available) were also performed. Readers offered a final interpretation of normal, abnormal, or indeterminate/borderline for each VSI examination, chest X-ray, and CT.ResultsOperators scanned 102 subjects (0–89 years old) for analysis. Lung VSI showed a sensitivity of 93% and a specificity of 91% for an abnormal chest X-ray and a sensitivity of 100% and a specificity of 93% for a clinical diagnosis of pneumonia. When any cases with an indeterminate rating on chest X-ray or ultrasound were excluded (n=38), VSI lung ultrasound showed 92% agreement with chest X-ray (Cohen’s κ 0.83 (0.68 to 0.97, p<0.0001)). Among cases with CT (n=21), when any ultrasound with an indeterminate rating was excluded (n=3), there was 100% agreement with VSI.ConclusionLung VSI performed by previously inexperienced ultrasound operators after brief training showed excellent agreement with chest X-ray and high sensitivity and specificity for a clinical diagnosis of pneumonia. Blinded readers were able to identify other respiratory diseases including pulmonary oedema and pleural effusion. Deployment of lung VSI could benefit the health of the global community.