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Sarcoidosis is an antigen-mediated chronic multisystem inflammatory disease characterized by the formation of non-caseating epithelioid granulomas that disrupt tissue architecture, trigger fibrosis, and precipitate organ dysfunction. Cardiac involvement portends a poorer prognosis, presenting with conduction disease, heart failure, pulmonary hypertension, valvular disease, pericardial effusions, tamponade, ventricular arrhythmias, and sudden death. A 75-year-old man with a history of heart failure with mildly reduced ejection fraction, suspected ocular sarcoidosis, monoclonal gammopathy of uncertain significance and 6 months of exertional dyspnea and lower extremity edema presented with acute-on-chronic volume overload. His labs were notable for elevated inflammatory markers. Cardiac MRI showed pericardial thickening, complex pericardial fluid. The differential diagnosis included tamponade, restrictive cardiomyopathy or constrictive pericarditis secondary to paraproteinemia, viral/idiopathic, vasculitis, tuberculosis, lupus, or sarcoidosis. Simultaneous right and left heart catheterization was consistent with constriction. The patient underwent surgical pericardiectomy. The pathology was consistent with sarcoidosis and intraoperative cultures grew Propionibacterium acnes. This case reviews the mechanism and diagnosis of sarcoidosis, the diagnosis of constrictive pericarditis, and a possible role for P. acnes in the development of sarcoidosis. Constrictive pericarditis is a rare presentation of cardiac sarcoidosis, particularly when the only other suspected involvement of sarcoidosis is uveitis. P. acnes has been associated with granuloma formation in sarcoid, although the mechanism is still unknown. P. acnes may represent a potential therapeutic target once a mechanism is further elucidated.

Rationale: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. Objective: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. Methods and Results: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6c LO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk ; reciprocal transplantation of Mertk +/+ marrow into Mertk −/− mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. Conclusions: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Rationale:Various types of viable stem cells have been reported to result in modest improvement in cardiac function after acute myocardial infarction. The mechanisms for improvement from different stem cell populations remain unknown. Objective:To determine whether irradiated (nonviable) embryonic stem cells (iESCs) improve postischemic cardiac function without adverse consequences. Methods and Results:After coronary artery ligation-induced cardiac infarction, either conditioned media or male murine or male human iESCs were injected into the penumbra of ischemic myocardial tissue of female mice or female rhesus macaque monkeys, respectively. Murine and human iESCs, despite irradiation doses that prevented proliferation and induced cell death, significantly improved cardiac function and decreased infarct size compared with untreated or media-treated controls. Fluorescent in situ hybridization of the Y chromosome revealed disappearance of iESCs within the myocardium, whereas 5-bromo-2′-deoxyuridine assays r...

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.

Rationale: Fibrosis is an important structural contributor to formation of atrial fibrillation (AF) substrate in heart failure. Transforming growth factor-β (TGF-β) signaling is thought to be intricately involved in creation of atrial fibrosis. Objective: We hypothesized that gene-based expression of dominant-negative type II TGF-β receptor (TGF-β-RII-DN) in the posterior left atrium in a canine heart failure model will sufficiently attenuate fibrosis-induced changes in atrial conduction and restitution to decrease AF. Because AF electrograms are thought to reflect AF substrate, we further hypothesized that TGF-β-RII-DN would lead to increased fractionation and decreased organization of AF electrograms. Methods and Results: Twenty-one dogs underwent injection+electroporation in the posterior left atrium of plasmid expressing a dominant-negative TGF-β type II receptor (pUBc-TGFβ-DN-RII; n=9) or control vector (pUBc- LacZ ; n=12), followed by 3 to 4 weeks of right ventricular tachypacing (240 bpm). Compared with controls, dogs treated with pUBC-TGFβ-DN-RII demonstrated an attenuated increase in conduction inhomogeneity, flattening of restitution slope and decreased duration of induced AF, with AF electrograms being more fractionated and less organized in pUBc-TGFβ-DN-RII versus pUBc- LacZ dogs. Tissue analysis revealed a significant decrease in replacement/interstitial fibrosis, p-SMAD2/3 and p-ERK1/2. Conclusions: Targeted gene-based reduction of TGF-β signaling in the posterior left atrium—with resulting decrease in replacement fibrosis—led to beneficial remodeling of both conduction and restitution characteristics of the posterior left atrium, translating into a decrease in AF and increased complexity of AF electrograms. In addition to providing mechanistic insights, this data may have important diagnostic and therapeutic implications for AF.

Background Mobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology. Objective To determine if phagocytic receptor MERTK is expressed in human hearts and to elucidate key sequential steps and phagocytosis efficiency of dying adult cardiomyocytes, by macrophages. Results In infarcted human hearts, expression profiles of the phagocytic receptor MER-tyrosine kinase (MERTK) mimicked that found in experimental ischemic mouse hearts. Electron micrographs of myocardium identified MERTK signal along macrophage phagocytic cups and Mertk −/− macrophages contained reduced digested myocyte debris after myocardial infarction. Ex vivo co-culture of primary macrophages and adult cardiomyocyte apoptotic bodies revealed reduced engulfment relative to resident cardiac fibroblasts. Inefficient clearance was not due to the larger size of myocyte apoptotic bodies, nor were other key steps preceding the formation of phagocytic synapses significantly affected; this included macrophage chemotaxis and direct binding of phagocytes to myocytes. Instead, suppressed phagocytosis was directly associated with myocyte-induced inactivation of MERTK, which was partially rescued by genetic deletion of a MERTK proteolytic susceptibility site. Conclusion Utilizing an ex vivo co-cultivation approach to model key cellular and molecular events found in vivo during infarction, cardiomyocyte phagocytosis was found to be inefficient, in part due to myocyte-induced shedding of macrophage MERTK. These findings warrant future studies to identify other cofactors of macrophage–cardiomyocyte cross-talk that contribute to cardiac pathophysiology.

This paper uses phospholipase Cepsilon as a model to demonstrate that lipids can act as ligands to bind to specific motifs and regulate protein activity via allosteric effects. Phospholipids such as phosphatidic acid and free fatty acids such as arachidonate are potent activators of PLCepsilon, increasing the rate of PI hydrolysis by 8-fold and 50-fold, respectively. The mechanism appears to be a reduction of K(m), as the substrate dependence curve is shifted to the left and K(m) is reduced 10-fold. The regulation of PLCepsilon by lipids appears to be physiologic, as reconstitution or cotransfection of either cPLA(2) or PLD with PLCepsilon leads to activation of phosphodiesterase activity. Additionally, TSA-201 cells transfected with PLCepsilon and fed arachidonic acid complexed with BSA had increased (4-5-fold) hydrolysis of polyphosphoinositides. This study demonstrates the ability of lipids to act as potent and direct mediators of protein function and identifies cross talk between different classes of phospholipase (PLD and PLA(2) with PLC) mediated via lipid products.

Infection of human erythrocytes by the malarial parasite, Plasmodium falciparum, results in complex membrane sorting and signaling events in the mature erythrocyte. These events appear to rely heavily on proteins resident in erythrocyte lipid rafts. Over the past five years, we and others have undertaken a comprehensive characterization of major proteins present in erythrocyte detergent-resistant membrane lipid rafts and determined which of these proteins traffic to the host-derived membrane that bounds the intraerythrocytic parasite. The data suggest that raft association is necessary but not sufficient for vacuolar recruitment, and that there is likely a mechanism of active uptake of a subset of erythrocyte detergent-resistant membrane proteins. Of the ten internalized proteins, few have been evaluated for a role in malarial entry. The β2-adrenergic receptor and heterotrimeric G protein Gs signaling pathway proteins regulate invasion. The implications of these differences are discussed. In addition, the...

One of the hallmarks of mammalian nephrogenesis includes a mesenchymal-epithelial transition that is accomplished by intercalation of the ureteric bud, an epithelium-lined tubelike structure, into an undifferentiated mesenchyme, and the latter then undergoes an inductive transformation and differentiates into an epithelial phenotype. At the same time, the differentiating mesenchyme reciprocates by inducing branching morphogenesis of the ureteric bud, which forms a treelike structure with dichotomous iterations. These reciprocal inductive interactions lead to the development of a functioning nephron unit made up of a glomerulus and proximal and distal tubules. The inductive interactions and differentiation events are modulated by a number of transcription factors, protooncogenes, and growth factors and their receptors, which regulate the expression of target morphogenetic modulators including the ECM, integrin receptors, and cell adhesion molecules. These target macromolecules exhibit spatiotemporal and stage-specific developmental regulation in the metanephros. The ECM molecules expressed at the epithelial-mesenchymal interface are perhaps the most relevant and conducive to the paracrine-juxtacrine interactions in a scenario where the ligand is expressed in the mesenchyme while the receptor is located in the ureteric bud epithelium or vice versa. In addition, expression of the target ECM macromolecules is regulated by matrix metalloproteinases and their inhibitors to generate a concentration gradient at the interface to further propel epithelial-mesenchymal interactions so that nephrogenesis can proceed seamlessly. In this review, we discuss and update our current understanding of the role of the ECM and related macromolecules with respect to metanephric development.

Imprinted mesodermal specific transcript ( MEST ) and H19 genes in renal development and diabetes. Background Imprinted genes, mesodermal specific cDNA or transcript ( MEST ) and H19 , are implicated in peri-implantation embryogenesis, and their expression was assessed in embryonic kidneys undergoing glucose-induced dysmorphogenesis. Methods MEST and H19 mRNA expression was assessed by Northern blot analysis in embryonic kidneys of mice harvested at day 15 to day 19 of gestation and of 1-week-old mice obtained from hyperglycemic mothers. A full-length mouse MEST cDNA was isolated, subcloned into an expression vector, a recombinant protein prepared and an antibody raised; the latter was used to assess protein expression by immunoprecipitation and immunofluorescence microscopy in day 13 metanephric explants subjected to high glucose ambience. Also, MEST mRNA expression was assessed in high d glucose–treated explants by competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses and by in situ tissue autoradiography. Results A high expression of MEST and H19 with respective transcript size of ∼2.7 and ∼2.4 kb was observed in fetal kidneys, and their expression decreased during the successive stages of gestation and was undetectable in the postnatal period. At day 13, the MEST mRNA was expressed in the mesenchyme, while H19 was expressed in the ureteric bud branches and epithelial elements of the metanephros. Their expression decreased with progression of gestation. By competitive RT-PCR and Northern blot and in situ autoradiographic analyses, both MEST and H19 expressions decreased in day 13 explants treated with high glucose and in the kidneys of fetuses obtained from diabetic mothers. The MEST protein expression was observed in the metanephric epithelial elements and ureteric bud branches instead of in the mesenchyme, and its expression decreased in glucose-treated dysmorphogenetic explants, as assessed by immunofluorescence and immunoprecipitation methods. Conclusion MEST and H19 imprinted genes are strategically located in the mammalian embryonic metanephros. They are developmentally regulated and their concomitant decreased expression in high glucose ambience or diabetic state did not follow the prevailing dogma of reciprocal inactivation/activation of imprinted genes, and such a decrease may be responsible for the perturbed epithelial:mesenchymal interactions leading to dysmorphogenesis of the mammalian metanephros.

Three families of phospholipase C (PI-PLCbeta, gamma, and delta) are known to catalyze the hydrolysis of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) to generate the second messengers inositol 1,4,5 trisphosphate and diacylglycerol, leading to a cascade of intracellular responses that result in cell growth, cell differentiation, and gene expression. Here we describe the founding member of a novel, structurally distinct fourth family of PI-PLC. PLCepsilon not only contains conserved catalytic (X and Y) and regulatory domains (C2) common to other eukaryotic PLCs, but also contains two Ras-associating (RA) domains and a Ras guanine nucleotide exchange factor (RasGEF) motif. PLCepsilon hydrolyzes PIP(2), and this activity is stimulated selectively by a constitutively active form of the heterotrimeric G protein Galpha(12). PLCepsilon and a mutant (H1144L) incapable of hydrolyzing phosphoinositides promote formation of GTP-Ras. Thus PLCepsilon is a RasGEF. PLCepsilon, the mutant H1144L, and the isolated GEF domain activate the mitogen-activated protein kinase pathway in a manner dependent on Ras but independent of PIP(2) hydrolysis. Our findings demonstrate that PLCepsilon is a novel bifunctional enzyme that is regulated by the heterotrimeric G protein Galpha(12) and activates the small G protein Ras/mitogen-activated protein kinase signaling pathway.

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

OBJECTIVES We sought to determine the relationship of delayed hyperenhancement by contrast magnetic resonance imaging (MRI) to viable and nonviable myocardium within the region at risk throughout infarct healing. BACKGROUND The relationship of delayed MRI contrast enhancement patterns to injured but viable myocardium within the ischemic bed at risk has not been established. METHODS We compared in vivo and ex vivo MRI contrast enhancement to histopathologic tissue sections encompassing the entire left ventricle in dogs (n = 24) subjected to infarction with (n = 12) and without (n = 12) reperfusion at 4 h, 1 day, 3 days, 10 days, 4 weeks and 8 weeks. In vivo MR imaging was performed 30 min after contrast injection. RESULTS The sizes and shapes of in vivo myocardial regions of elevated image intensity (828 ± 132% of remote) were the same as those observed ex vivo (241 slices, r = 0.99, bias = 0.05 ± 1.6% of left ventricle [LV]). Comparison of ex vivo MRI to triphenyltetrazolim chloride–stained sections demonstrated that the spatial extent of hyperenhancement was the same as the spatial extent of infarction at every stage of healing (510 slices, lowest r = 0.95, largest bias = 1.7 ± 2.9% of LV). Conversely, hyperenhanced regions were smaller than the ischemic bed at risk defined by fluorescent microparticles at every stage of healing (239 slices, 35 ± 24% of risk region, p CONCLUSIONS Delayed contrast enhancement by MRI distinguishes between viable and nonviable regions within the myocardium at risk throughout infarct healing.

The inositol phosphate hydrolyzing activity of human phospholipase Cδ1 (PLCδ1) is markedly inhibited when the enzyme is coexpressed with the human heart G h /transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLCδ1 in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLCδ1. Nucleotides (GTP > GDP > ATP > GMP = ADP, in order of decreasing efficiency) interfered with the formation of the PLCδ1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: TG slow + GTP ⇆ [TG:GTP] fast . Altogether, our findings indicate that GTP controls PLCδ1 activity by releasing this protein from an inhibitory association with G h /transglutaminase.

The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.

Background Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal large B-cell lymphoma characterized by the selective growth of large lymphoma cells within smaller vessel lumina. With less than 500 reported cases, little is understood about this extranodal B-cell lymphoma. This entity is considered widely underdiagnosed due to its nonspecific clinical presentation, often not detected until autopsy. Cytogenetic and immunophenotypic analyses of IVLBCL cases have revealed the presence of numerous chromosomal aberrations and the non-germinal center (post-germinal center) activated B-cell phenotype. Chromosomal abnormalities and clonal immunoglobulin rearrangements have been reported, however, very few cases have been studied. Methods We performed an IRB-approved retrospective review of the electronic medical records in our institution from January 2007 to January 2015 and identified five cases of IVLBCL. Morphologic evaluation, immunohistochemical (IHC) stains and PCR analysis for clonality was performed using primers specific for the immunoglobulin heavy (IgH) and light chains (IgK and IgL). IHC was used to characterize the neoplastic cells into germinal center B cell (GCB) and non-GCB phenotypes according to the Hans criteria. Results We identified five patients with the pathologic diagnosis of IVLBCL, three of whom were autopsy cases and two that were living patients referred for consultation. The clinical and pathologic features of these cases are summarized in Table 1. All three autopsy cases had multiorgan involvement by IVLBCL evidenced by CD20 positivity (see Image 1) and with the diagnosis made for the first time post-mortem. The two living patients had an initial presentation of rash and skin involvement. Brain involvement was present in two cases; in the first, which was an autopsy case, it presented in a perivascular morphologic pattern, and in the second case, it presented six months following the intradermal diagnosis of IVLBCL and subsequent R-CHOP treatment, as an extravascular 4.5 cm mass-forming lesion. This patient has been disease-free since receiving autologous stem cell transplant. Of the four cases with available material for IHC, the neoplastic cells were of non-GCB phenotype for three cases and GCB phenotype for one case. Clonal IgH rearrangement was identified in one of the five cases. Clonal light chain rearrangements were identified in two cases. Conclusions IVLBCL is an elusive diagnosis and more than half of our cases were diagnosed first time post-mortem. Similar to literature described, this is a highly aggressive lymphoma. The two patients in our series with mostly skin involvement appear to have a better prognosis. We also observed IVLBCL may not always present intravascularly, particularly in the brain, where it may present as perivascular or even form extravascular mass lesions. PCR analysis could detect clonal immunoglobulin gene rearrangements. However, assessing heavy chain gene rearrangement alone is not sensitive. PCR of the immunoglobulin light chain, particularly IgK, is a useful technique to increase the sensitivity for detection of clonality for IVLBCL. However, PCR analysis for clonality is inderminate in two cases and this could be attributed to DNA degradation as well as low tumor volume. Future directions that will be explored include performing next generation sequencing on VDJ junctions for these cases. | Case | Age | Sex | Clinical Features | Organ Involvement by Histology | GCB vs. non-GCB | PCR | | ---- | --- | --- | ------------------------------------------------------------------------------------------------------------------------------------------- | ----------------------------------- | --------------- | ------------------------------------------------------------------------ | | 1 | 81 | M | Respiratory failure, splenomegaly, thrombocytopenia, coagulopathy. | Multiorgan | Non-GCB | IgH: Indeterminate IgL: Indeterminate IgK: Indeterminate | | 2 | 76 | F | Deceased; presented with encephalopathy. Bone marrow involvement present. | Multiorgan | Non-GCB | IgH: Indeterminate IgL: Indeterminate IgK: Clonal | | 3 | 61 | F | 6 month of progressive rash, weakness, SOB, fever. Presented 6 month later with 4.5 cm left frontal brain lymphoma. Currently in remission. | Skin initially. Subsequently brain. | GCB | IgH: Indeterminate IgL: Indeterminate IgK: Indeterminate | | 4 | 56 | F | Remote history of non-Hodgkins lymphoma who subsequently presented with rash. | N/A | N/A | IgH: Indeterminate IgL: Clonal IgK: Clonal | | 5 | 84 | M | Presented with 3 months of fevers, chills, new thrombocytopenia. | Multiorgan | Non-GCB | IgH: Clonal IgL: Clonal IgK: Clonal | Table 1. ![Figure 1.][1] Figure 1. Disclosures No relevant conflicts of interest to declare. [1]: pending:yes

alpha 2-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. alpha 2-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective alpha 2-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B, and alpha 2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with 35S-labeled RNA probes to map the distribution of alpha 2-adrenoceptor subtype gene expression in rat brain. alpha 2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. alpha 2B mRNA was observed only in the thalamus. alpha 2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the alpha 2-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of alpha 2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of alpha 2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the alpha 2-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.

The dynamic component of bladder outlet obstruction caused by benign prostatic hyperplasia (BPH) is regulated by alpha 1 adrenergic receptors (alpha 1-AR) located in the prostatic stroma. Recently two alpha 1-AR subtypes (alpha 1A, alpha 1B) have been identified in the human prostate by both functional and pharmacological assays. However, the presence of the alpha 1C subtype has not been evaluated, presumably due to the lack of availability of selective ligands for this receptor subtype. We have used molecular techniques to investigate the mRNA expression of all three alpha 1-AR subtypes in the human prostate. RNA extracted from the prostate gland of 15 patients was used in ribonuclease protection assays to identify the expression of three alpha 1-AR subtype mRNAs. Quantitative solution hybridization assays further identify the predominant subtype of alpha 1-AR mRNA to be the alpha 1C, which represents approximately 70% of the total alpha 1-AR mRNA in the human prostate. Furthermore, in situ hybridization localizes the alpha 1C AR mRNA predominantly to the stromal compartment. The identification of a predominant alpha 1-AR mRNA in human prostate identifies a potential need for subtype selective pharmaceutical agents. These agents could be very important clinically in the treatment of diseases such as BPH.

In membranes prepared from Cos-7 or HeLa cells expressing one of three individual cloned alpha 1-adrenoceptor subtypes, competition with 2-[(beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-tetralone ([125I]HEAT) by the selective compounds [+]-niguldipine, 5-methyl-urapidil, and benoxathian reveals high affinity for the cloned alpha 1C-adrenoceptor subtype and low affinity for both the cloned alpha 1A-adrenoceptor and alpha 1B-adrenoceptor. Competition with [125I]HEAT by spiperone revealed high affinity for the cloned alpha 1C-adrenoceptor, intermediate affinity for the cloned alpha 1B-adrenoceptor, and low affinity for the cloned alpha 1A-adrenoceptor. Combining pharmacological properties previously described for alpha 1-adrenoceptor subtypes in rat membranes and here described from cloned receptors, these data suggest the existence of a fourth distinct alpha 1-adrenoceptor subtype.

This study investigated the effects on cardiomyocyte differentiation of embryonic stem cells by the overexpression of the transcription factor, Pitx2c, and examined the effects of transplantation of these differentiated cells on cardiac function in a mouse model of myocardial infarction. Pitx2c overexpressing embryonic stem cells were characterized for cardiac differentiation by immunocytochemistry, RNA analysis, and electrophysiology. Differentiated cells were transplanted by directed injection into the infarcted murine myocardium and functional measurements of blood pressure, contractility, and relaxation were performed. Histochemistry and FISH analysis performed on these mice confirmed the engraftment and cardiac nature of the transplanted cells. Pitx2c overexpressing embryonic stem cells robustly differentiated into spontaneously contracting cells which acquired cardiac protein markers and exhibited action potentials resembling that of cardiomyocytes. These cells could also be synchronized to an external pacemaker. Significant improvements (P < 0.01) in blood pressure (56%), contractility (57%), and relaxation (59%) were observed in infarcted mice with transplants of these differentiated cells but not in mice which were transplanted with control cells. The Pitx2c overexpressing cells secrete paracrine factors which when adsorbed onto a heparinated gel and injected into the infarcted myocardium produce a comparable and significant (P < 0.01) functional recovery. Pitx2c overexpression is a valuable method for producing cardiomyocytes from embryonic stem cells, and transplantation of these cardiomyocytes into infracted myocardium restores cardiac function through multiple mechanisms.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.

Sympathetic nervous system development depends upon many factors that mediate neuron migration, differentiation and survival. Target tissue-derived nerve growth factor (NGF) signaling-induced gene expression is required for survival, differentiation and target tissue innervation of post-migratory sympathetic neurons. However, the transcriptional regulatory mechanisms mediated by NGF signaling are very poorly defined. Here, we identify Egr3, a member of the early growth response (Egr) family of transcriptional regulators, as having an important role in sympathetic nervous system development. Egr3 is regulated by NGF signaling and it is expressed in sympathetic neurons during development when they depend upon NGF for survival and target tissue innervation. Egr3-deficient mice have severe sympathetic target tissue innervation abnormalities and profound physiological dysautonomia. Unlike NGF, which is essential for sympathetic neuron survival and for axon branching within target tissues, Egr3 is required for normal terminal axon extension and branching, but not for neuron survival. The results indicate that Egr3 is a novel NGF signaling effector that regulates sympathetic neuron gene expression required for normal target tissue innervation and function. Egr3-deficient mice have a phenotype that is remarkably similar to humans with sympathetic nervous system disease, raising the possibility that it may have a role in some forms of human dysautonomia,most of which have no known cause.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

A novel alpha 1-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypeptide. The structure is similar to that of the other adrenergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven-membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alpha 1B-adrenergic receptor being approximately 72% within the presumed membrane-spanning domains. Localization on different human chromosomes provides evidence that the bovine cDNA is distinct from the hamster alpha 1B-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed 10-fold higher affinity for the alpha 1-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline as compared with the alpha 1B receptor, results similar to pharmacologic binding properties described for the alpha 1A receptor. Despite these similarities in pharmacological profiles, the bovine alpha 1-adrenergic receptor is sensitive to inhibition by the alkylating agent chloroethylclonidine unlike the alpha 1A-adrenergic receptor subtype. In addition, a lack of expression in tissues where the alpha 1A subtype exists suggests that this receptor may actually represent a novel alpha 1-adrenergic receptor subtype not previously appreciated by pharmacological criteria.

Introduction Hereditary amyloidodis is a rare disease process with a propensity to cause polyneuropathies, autonomic dysfunction, and restrictive cardiomyopathy. It is transmitted in an autosomal dominant manner, with disease onset usually in the 20s-40s. The most common hereditary amyloidogenic protein, transthyretin, is synthesized in the liver and lies on Chromosome 18. Over 80 amyloidogenic transthyretin mutations have been described, the majority of which are neuropathic and hence the common name, Familial Amyloidotic Polyneuropathy. Until 1990, the disease was intractable with a 5–15 year survival after diagnosis. The prognosis changed after the implementation of orthotropic liver transplantation as a treatment strategy which halts the synthesis of amyloidogenic transthyretin. This has now has been performed over 1300 times in 67 centers. Case presentation We describe the case of a man of Irish ancestry with Familial Amyloidotic Polyneuropathy and no clinical history of cardiac involvement. Shortly after orthotropic liver transplantation, he developed congestive heart failure. He was subsequently diagnosed with an accelerating post-transplant restrictive cardiomyopathy due to amyloid infiltration. Conclusion A liver transplant induced cardiomyopathy in Familial Amyloidotic Polyneuropathy can be observed in patients without any history of cardiac symptoms. All patients with Familial Amyloidotic Polyneuropathy should be followed after transplantation to assess for a deterioration in cardiac function.

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non–receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP2) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP2, although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.

Background Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum. Methods and Findings We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection. Conclusions Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials., Erythrocyte G protein signaling is needed for intracellular malarial parasite proliferation and thus may present a novel antimalarial target., Editors' Summary Background. New drugs for treatment of malaria are urgently needed, because the malaria parasite has evolved resistance against virtually all types of commonly used drugs. When a person is bitten by a malaria-infected mosquito, the parasite first infects the person's liver cells before going on to infect red blood cells, where the parasites multiply and develop into a parasite stage called a schizont. The red blood cells then burst and release more schizonts into the bloodstream; it is this “blood stage” of infection in humans that causes the symptoms of disease. Therefore efforts to develop new drugs against malaria often focus on this “blood stage” of infection. One strategy for developing new drugs is termed the “host-targeted” approach. This means that rather than trying to block processes occurring within the parasite itself, a drug can be developed which blocks processes within the person's red blood cells, and which would otherwise be needed for the parasite to complete its life cycle. It will be difficult for malaria parasites to evolve resistance to such a drug, because changes in a person's red blood cells occur much more slowly than in the parasites themselves. Why Was This Study Done? This research group has been studying a set of molecular processes within human red blood cells which seemed to be required for entry of malaria parasites into the cells. They wanted to get a better understanding of those processes and, specifically, to find out whether it would be possible to use particular molecules to block those processes, and by doing so to prevent malaria parasites from entering and multiplying within red blood cells. In particular, when the malaria parasites invade the red blood cell, they form membranes around the red blood cell, containing lipids and proteins “hijacked” from the red blood cell membrane. These researchers already knew that two particular proteins were hijacked in this way; the β2-adrenergic receptor (β2-AR) and heterotrimeric G protein (Gs). These two proteins act together to pass messages across the surface of the membrane to inside the cell. Small molecules could be used to block signaling through β2-AR and Gs, and therefore potentially to provide a new way of preventing malaria parasites from entering red blood cells and multiplying within them. What Did the Researchers Do and Find? Firstly, the researchers made red blood cell “ghosts” in which to study these molecular processes. This meant that they took fresh red blood cells from healthy human volunteers, burst them to remove half the contents and loaded them with markers and other cargoes before resealing the membranes of the cell. These resealed markers and cargoes allowed them to see what was happening inside the cells. Malaria parasites were able to invade these ghosts normally and multiply within them. When the researchers introduced a specific peptide (a molecule consisting of a short series of amino acids), they found that it blocked Gs signaling within the ghosts. This peptide also prevented malaria parasites from developing inside the ghosts. Therefore, they concluded that Gs signaling inside the red blood cell was important for the parasite life cycle. The researchers then examined a drug called propranolol which is already known to act on Gs signaling and which is commonly prescribed for high blood pressure. This drug also blocked development of malaria parasites inside the ghosts when used at a particular concentration. Finally, the researchers studied the effect of giving propranolol, along with other antimalarial drugs, to human malaria parasites in a culture dish and to mice injected with a malaria parasite that infects rodents. In these experiments, adding propranolol reduced the amount of other “parasite-targeted” drugs that were needed to effectively treat malarial infection in tissue culture and in mice. What Do These Findings Mean? Showing that the Gs signaling pathway is important for the malaria parasite's life cycle opens up new possibilities for drug development. Specifically, propranolol (which is already approved for treatment of high blood pressure and other conditions) might itself provide a new candidate therapy, either alone or in combination with existing drugs. These combinations would first, however, need to be tested in human clinical trials, perhaps by seeing whether they have antimalarial activity in people who have not responded to existing antimalarial drugs. Since it acts to lower blood pressure, which can already be low in some people with malaria, there are some concerns that propranolol might not be a suitable drug candidate for use, especially with existing antimalarial drugs that also reduce blood pressure. However, other molecules which block Gs signaling could be tested for activity against malaria should propranolol prove not to be an ideal drug candidate. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030528. The World Health Organization publishes a minisite containing links to information about all aspects of malaria worldwide, including treatment, prevention, and current programs for malaria control Medicines for Malaria Venture is a collaboration between public and private organizations (including the pharmaceutical industry) that aims to fund and manage the development of new drugs for treatment and prevention of malaria Wikipedia entries for drug discovery and drug development (Wikipedia is an internet encyclopedia that anyone can edit)

Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte β2-adrenergic receptor and heterotrimeric guanine nucleotide–binding protein (Gαs) regulated the entry of the human malaria parasite Plasmodium falciparum . Agonists that stimulate cyclic adenosine 3′,5′-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Gαs protein function reduced parasitemia in P. falciparum cultures in vitro, and β-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte β2-adrenergic receptor and Gαs may regulate malarial infection across parasite species.

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.

Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.

This chapter focuses on adrenergic receptors, which mediate important cardiovascular effects, including regulation of blood pressure, myocardial contractile rate (chronotropy), myocardial force (inotropy), and myocardial relaxation (lusitropism). The nine individual subtypes of ARs belong to three distinct receptor families: α1, α2, and β ARs. Each receptor has its own unique structural, biochemical, pharmacolgical, and physiological properties. In the cardiovascular system, α1-ARs are an important regulator of vascular tone and hence, blood pressure. They may also have effects on atrial conduction and play a role in the development of cardiac arrythmias. Hormone or agonist binding to members of the three families of adrenergic receptor α1, α2, and β initiates a cascade of molecular events, which includes coupling of the receptor with a heterotrimeric guanine nucleotide regulatory protein (G-protein) and subsequent interaction of the G-protein with various effector molecules. This signaling pathway is itself regulated dynamically, resulting in a dampening of agonist responses over time, a process referred to as desensitization. A family of G-protein receptor kinases plays an important role in uncoupling the receptor from the G-protein, resulting in desensitization.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.