Novel mechanism of plasma prekallikrein (PK) activation by vascular smooth muscle cells: evidence of the presence of PK activator.

The contribution of plasma prekallikrein (PK) to vascular remodeling is becoming increasingly recognized. Plasma PK is activated when the zymogen PK is digested to an active enzyme by activated factor XII (FXII). Here, we present our findings that vascular smooth muscle cells (VSMC) activate plasma PK in the absence of FXII. Extracted plasma membrane and cytosolic fractions of VSMCs activate PK, but the rate of PK activation was greater by the membrane fraction. FXII neutralizing antibody did not affect PK activation by extracted proteins of VSMCs. VSMC PKA was inhibited by the serine protease inhibitors such as aprotinin, phenylmethylsulfonyl fluoride, leupeptin and CTI with CI50 of 0.78 µM, 1 mM, 3.13 µM and 40 nM on the cultured cells, respectively. No inhibition of PK activation by cysteine, aspartic acid, and metalloprotease inhibitors was observed. This is the first report of the presence of an intrinsic PKA in VSMC. Considering that VSMCs are normally separated from the circulating blood by endothelial cells, direct PK activation by VSMCs may play a role in disease states like diabetes, hyperlipidemia or hypertension where the endothelial layer is damaged.

Regulation of tyrosine kinase cascades by G-protein-coupled receptors.

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.

Phosphatidylinositol-glycan anchors of membrane proteins: potential precursors of insulin mediators.

BC3H1 myocytes release membrane-bound alkaline phosphatase to the incubation medium upon stimulation with insulin, following a time course that is consistent with the generation of dimyristoylglycerol and the appearance of a putative insulin mediator in the extracellular medium. The use of specific blocking agents shows, however, that alkaline phosphatase release and dimyristoylglycerol production are independent processes and that the blockade of either event inhibits the production of insulin mediator. These experiments suggest a new model of insulin action.

Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors.

The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.

Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy.

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Augmented mitogenic responsiveness to epidermal growth factor in murine fibroblasts that overexpress pp60c-src.

C3H10T1/2 murine fibroblasts overexpressing chicken pp60c-src showed a two- to fivefold enhanced incorporation of [3H]thymidine into DNA in response to epidermal growth factor (EGF) relative to that of the parent line. No difference in growth characteristics, number and affinity of EGF receptors, or hormone potency was attributable to c-src overexpression. These results suggest that pp60c-src may interact with the mitogenic signal transduction pathway of EGF in some event distal to hormone binding.

Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity.

Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.

Regulator of G protein signaling 2 is a key regulator of pancreatic ß-cell mass and function.

Pancreatic ß-cell death and dysfunction contributes to the pathogenesis of both type 1 and type 2 diabetes. We aimed to examine whether the regulator of G protein signaling protein 2 (RGS2), a multifunctional inhibitor of G protein-coupled receptor (GPCR) signaling, impacts ß-cell death and function. Metabolic phenotypes, ß-cell secretory function, and glucose and insulin tolerance were measured in RGS2 knockout (RGS2-/-) mice and their wild-type (RGS2+/+) littermate controls. ß-Cell death was evaluated in RGS2-knockdown and -overexpressing ß cells and RGS2-/- islets by flow cytometry, western blot, ELISA, TUNEL staining, and apoptosis RT2 profiler PCR array analysis. ß-Cell mass was evaluated in pancreases from RGS2-/- and RGS2+/+ mice at 1 day, 4 weeks, and 25 weeks of age. Our data show that RGS2-/- islets secreted more insulin than RGS2+/+ islets when challenged with glucose or exendin-4. RGS2-knockdown cells are susceptible to hypoxia induced cell death while RGS2-overexpressing cells are protected from cell death. Depletion of RGS2 in islets alters expression of apoptosis-related genes and RGS2-/- islets are prone to apoptosis compared with RGS2+/+ islets. Ultimately, excessive insulin secretion and increased ß-cell apoptosis contributed to a 70% reduction in pancreatic ß-cell mass in RGS2-/- mice compared with RGS2+/+ mice at 25 weeks of age. RGS2 has critical roles in maintaining pancreatic ß-cell mass via modulating ß-cell function and apoptosis. It may serve as a druggable target to help prevent pancreatic ß-cell loss in the treatment of diabetes.

Insulin and phorbol ester induce distinct phosphorylations of pp60c-src in the BC3H-1 murine myocyte cell line.

Insulin and phorbol esters have been shown to produce similar, non-additive metabolic effects in BC3H-1 murine myocytes. Recently, it has been demonstrated that insulin stimulation of these cells increases production of diacylglycerol, a known activator of protein kinase C (PK-C). To determine if insulin stimulation results in the activation of PK-C, we have examined the effects of insulin and the tumor promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the phosphorylation of a known PK-C substrate in vivo, the cellular proto-oncogene product, pp60c-src. Differentiated BC3H-1 monocytes showed an approximate twofold elevation in the [32P] content of pp60c-src following stimulation with insulin or TPA for 20 min, with no detectable change in the level of immunoprecipitable c-src protein. The enhanced phosphorylation in response to each agent localized to serine residues in the amino terminal 16 kD staphylococcal V8 proteolytic fragment. Tryptic phosphopeptide analysis revealed that TPA stimulation resulted in an approximate 18-fold increase in phosphorylation of the serine 12-containing tryptic fragment. Insulin stimulation, however, resulted in an approximate 10-fold increase in phosphorylation of the serine 17-containing tryptic fragment with little or no accompanying increase in serine 12 phosphorylation. In cells exposed to high concentrations of TPA for 16 h to deplete PK-C activity, insulin, but not TPA, stimulated phosphorylation of pp60c-src. These data suggest that insulin and phorbol ester induce phosphorylation of pp60c-src by distinct protein kinases.

New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades.

Activation of classical second messenger cascades cannot fully explain the recently appreciated roles of heptahelical, or G-protein coupled receptors (GPCRs), in stimulation of mitogen activated protein kinase (MAPK) cascades. Rather, several distinct signaling mechanisms appear to contribute to GPCR-mediated MAPK activation. These include transactivation of the Epidermal Growth Factor Receptor (EGFR) via the autocrine/paracrine release of EGF-like ligands at the cell surface and scaffolding of MAPK cascades. A significant advance in the understanding of how GPCRs activate MAPK cascades is the discovery that beta-arrestin, a protein well known for its roles in both receptor desensitization and internalization, serves as a scaffolding protein for at least two GPCR stimulated MAPK cascades, the extracellular signal regulated kinase (ERK) cascade and the c-jun N-terminal kinase 3 (JNK3) cascade. Together, these novel mechanisms of GPCR-mediated MAPK regulation may permit GPCRs in specific situations to control the temporal and spatial activity of MAPKs and thereby determine the consequences of GPCR stimulation with respect to transcriptional activation, cell proliferation and apoptosis.

Partial insulin resistance in the mouse BC3H-1 cell line: absent hexose-independent actions of insulin.

We have studied the regulation of glycogen metabolism by insulin in the insulin-sensitive nonfusing muscle cell line BC3H-1. The basal percentage of glycogen synthase I activity was not altered by insulin alone at any concentration, time of exposure, or age of cells tested. The addition of glucose or 2-deoxyglucose to the glucose- and serum-free incubation medium caused a 2-fold increase in glycogen synthase I activity over basal levels, and the effect was enhanced to 3-fold if insulin was added to the medium. Glycogen phosphorylase a activity was not altered by incubation in the presence of insulin, but was lowered by the addition of 2-deoxyglucose. This effect was also enhanced in the presence of insulin. The effect of exogenously added sugar occurred only if a 6-phosphorylatable hexose was used. The effect seen with 2-deoxyglucose was stable to Sephadex G-25 desalting, suggesting that activation of glycogen synthase was the result of a stable (covalent) modification of the enzyme. We were also able to demonstrate the presence of glucose-6-phosphate-activatable glycogen synthase phosphatase activity in the myocytes. The effect of 2-deoxyglucose in the presence or absence of insulin could be completely reversed by including cytochalasin B in the medium, suggesting that both the effect of hexose and the insulin enhancement of its effect were entirely dependent on carrier-mediated hexose uptake. Four insulin-mimetic agents, H2O2 Concanavalin A, Na orthovanadate, and antiinsulin receptor B2 serum, were also tested. Despite different mechanisms of action, each agent qualitatively mimicked insulin in the myocytes. All stimulated hexose transport, glucose incorporation into glycogen, and hexose-dependent activation of glycogen synthase in a manner not additive with insulin, but none increased basal glycogen synthase I activity in the absence of hexose. These results suggest that although insulin is capable of regulating glycogen metabolism both by increasing the uptake of sugar and by altering the activation state of glycogen synthase and phosphorylase, these effects are entirely due to the stimulation of hexose uptake, and hexose-independent actions of insulin are absent in BC3H-1 cells.

'Location, location, location': activation and targeting of MAP kinases by G protein-coupled receptors.

A growing body of data supports the conclusion that G protein-coupled receptors can regulate cellular growth and differentiation by controlling the activity of MAP kinases. The activation of heterotrimeric G protein pools initiates a complex network of signals leading to MAP kinase activation that frequently involves cross-talk between G protein-coupled receptors and receptor tyrosine kinases or focal adhesions. The dominant mechanism of MAP kinase activation varies significantly between receptor and cell type. Moreover, the mechanism of MAP kinase activation has a substantial impact on MAP kinase function. Some signals lead to the targeting of activated MAP kinase to specific extranuclear locations, while others activate a MAP kinase pool that is free to translocate to the nucleus and contribute to a mitogenic response.

Behavioral retardation in a macaque with autosomal trisomy and aging mother.

The social development of a female rhesus monkey (Macaca mulatta) was followed from the day of birth until her death, at age 32 months. The subject, born to an older mother, had an extra autosome (karyotype: 43, XX, +18), an affliction that came about spontaneously. MRI scans revealed that she was also hydrocephalic. Compared to 23 female monkeys growing up under identical conditions, the subject showed serious motor deficiencies, a dramatic delay in the development of social behavior, poorly established dominance relationships, and greater than usual dependency on mother and kin. The subject was well-integrated into the social group, however.

Achieving uniform dose with the use of a custom tissue compensator and a leveled beam for tangential breast fields.

In order to achieve uniform dose distribution in intact breast treatments, wedges can be employed. This paper will describe a custom compensator made from brass chips used in conjunction with a leveled beam and a custom "cast" to treat breast shapes that are less suited to a standard wedge set up. Materials and design, dosimetry, criteria, efficacy and results will be described.

Plantar pressure measurements during ambulation in weightbearing conventional short leg casts and total contact casts.

Total contact casts have been shown to be effective in healing plantar neuropathic ulcerations. The proposed mechanism of action is reduction of pressure over the ulcer during ambulation. However, there is little information to support this contention. Plantar pressure distribution was studied using standard short leg casts and total contact casts in normal feet. Both types of casts reduced forefoot pressure in study subjects. This occurred because of an increase in plantar surface area exposed to weightbearing forces. While increased force was shifted to the midfoot, there was no increase in pressure due to a corresponding increase in midfoot surface area exposed to this force. No significant difference was noted between standard short leg casts and total contact casts.

Functional signaling biases in G protein-coupled receptors: Game Theory and receptor dynamics.

Pharmacotherapeutic targeting of G protein-coupled receptors (GPCRs) is perhaps the most important field of drug design, as agents designed to control these receptors constitute more than half of the pharmacopeia. Initially GPCRs were considered to be unitary entities, possessing all of their potential functionality in their characteristic heptahelical core. Early models of the functional activity of GPCRs considered them to possess just a simple 'on' or 'off' status. Recent research however has allowed us to realize that GPCR functionality is dependent upon many other proteins outside of the heptahelical core, on the site of GPCR expression in a tissue or a microdomain in a cell, and, most importantly, on the formation of differential 'active' states preferentially coupled to specific signal transduction structures. The recognition of such signaling diversity has facilitated the ability to appreciate and identify ligands for GPCRs that demonstrate a bias towards one signaling form of a receptor to another. However while potentially increasing our ability for selective signal targeting, our approach to understanding the physiological ramifications of systemic signaling manipulation is underdeveloped. This explosion in the complexity of GPCR signaling is now becoming familiar territory to receptor biologists, yet the application of this knowledge to drug design is relatively limited. This review will attempt to outline potential pitfalls and unseen benefits of using signaling bias in therapeutic design as well as highlighting new applications such as Game Theory for uncovering new therapeutic applications for biased agonists.