Los receptores acoplados a proteínas G como blanco terapéutico / G protein-coupled receptors as therapeutic target

Resumen Los receptores son proteínas codificadas por el ADN, algunos de los cuales ya han sido cristalizados, lo que permite conocer los detalles de su estructura a nivel atómico y algunos aspectos de su función. Esta revisión se enfoca en los más diversos y abundantes, los receptores acoplados a la proteína G. Esta familia de receptores reconoce y media la acción de varios ligandos endógenos (hormonas, neurotransmisores, factores de crecimiento y hormonas locales) y también interviene en la patogenia de diversas enfermedades, por lo que son el blanco terapéutico de aproximadamente 30 a 40 % de los medicamentos que se emplean en la práctica clínica cotidiana y de diversas drogas ilegales. La cristalografía de rayos X es una de las herramientas clave que ha permitido observar la estructura de estos receptores en los aminoácidos que participan en esta interacción, lo que posibilita conocer el sitio de unión del ligando endógeno y de moléculas sintéticas que actúan sobre ellos para modular su acción. El modelado molecular es también una herramienta bioinformática computacional que apoya la investigación sobre la unión receptor-ligando, que hace posible el diseño y desarrollo de fármacos cada vez más específicos. A estos desarrollos se suman importantes cambios en los conceptos farmacodinámicos fundamentales.

Abstract Receptors are proteins coded by DNA, some of which have already been crystalized, thus allowing the details of their structure at the atomic level and some aspects of their function to be known. This review focuses on the most diverse and abundant family of receptors, G protein-coupled receptors. This family of receptors recognizes and mediates the action of several endogenous ligands (hormones, neurotransmitters, growth factors and local hormones) and also intervenes in the pathogenesis of various diseases, which is why they are targeted by approximately 30 to 40% of medications that are used in daily clinical practice and of various illegal drugs as well. X-ray crystallography is one of the essential tools that has allowed to observe the structure of these receptors in the amino acids that participate in this interaction, which allows to know the binding site of the endogenous ligand and of synthetic molecules that act on them to modulate their action. Molecular modeling or "docking" is also a computational bioinformatics tool that supports research on receptor-ligand binding, which allows the design and development of increasingly specific drugs. These developments have brought along significant changes in fundamental pharmacodynamic concepts.

Effect of pertussis toxin pretreated centrally on blood glucose level induced by stress

In the present study, we examined the effect of pertussis toxin (PTX) administered centrally in a variety of stress-induced blood glucose level. Mice were exposed to stress after the pretreatment of PTX (0.05 or 0.1 µg) i.c.v. or i.t. once for 6 days. Blood glucose level was measured at 0, 30, 60 and 120 min after stress stimulation. The blood glucose level was increased in all stress groups. The blood glucose level reached at maximum level after 30 min of stress stimulation and returned to a normal level after 2 h of stress stimulation in restraint stress, physical, and emotional stress groups. The blood glucose level induced by cold-water swimming stress was gradually increased up to 1 h and returned to the normal level. The intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with PTX, a Gi inhibitor, alone produced a hypoglycemia and almost abolished the elevation of the blood level induced by stress stimulation. The central pretreatment with PTX caused a reduction of plasma insulin level, whereas plasma corticosterone level was further up-regulated in all stress models. Our results suggest that the hyperglycemia produced by physical stress, emotional stress, restraint stress, and the cold-water swimming stress appear to be mediated by activation of centrally located PTX-sensitive G proteins. The reduction of blood glucose level by PTX appears to due to the reduction of plasma insulin level. The reduction of blood glucose level by PTX was accompanied by the reduction of plasma insulin level. Plasma corticosterone level up-regulation by PTX in stress models may be due to a blood glucose homeostatic mechanism.

Effect of D-glucose feeding on mortality induced by sepsis

Sepsis is the life-threatening response to infection which can lead to tissue damage, organ failure, and death. In the current study, the effect of orally administered D-glucose on the mortality and the blood glucose level induced by D-Galactosamine (GaLN)/lipopolysaccharide (LPS)-induced sepsis was examined in ICR mice. After various amounts of D-glucose (from 1 to 8 g/kg) were orally fed, sepsis was induced by injecting intraperitoneally (i.p.) the mixture of GaLN /LPS. Oral pre-treatment with D-glucose dose-dependently increased the blood glucose level and caused a reduction of sepsis-induced mortality. The oral post-treatment with D-glucose (8 g/kg) up to 3 h caused an elevation of the blood glucose level and protected the mortality observed in sepsis model. However, D-glucose post-treated at 6, 9, or 12 h after sepsis induction did not affect the mortality and the blood glucose level induced by sepsis. Furthermore, the intrathecal (i.t.) pretreatment once with pertussis toxin (PTX; 0.1 microg/5 ml) for 6 days caused a reduction of D-glucose-induced protection of mortality and hyperglycemia. Furthermore, once the hypoglycemic state is continued up to 6 h after sepsis initiated, sepsis-induced mortality could not be reversed by D-glucose fed orally. Based on these findings, it is assumed that the hypoglycemic duration between 3 and 6 h after the sepsis induction may be a critical time of period for the survival. D-glucose-induced protective effect against sepsis-induced mortality appears to be mediated via activating PTX-sensitive G-proteins in the spinal cord. Finally, the production of hyperglycemic state may be critical for the survival against the sepsis-induced mortality.

Regulation of Giα Protein Expression by Vasoactive Peptides in Hypertension: Molecular Mechanisms.

Guanine nucleotide regulatory proteins (G proteins) play a key role in the regulation of various signal transduction systems, including adenylyl cyclase/cAMP and phospholipase C (PLC)/phosphatidyl inositol (PI) turnover, which are implicated in the modulation of a variety of physiological functions, such as platelet functions, including platelet aggregation, secretion, and clot formation and cardiovascular functions, including arterial tone and reactivity. Several abnormalities in adenylyl cyclase activity, cAMP levels and G proteins have been shown to be responsible for the altered cardiac performance and vascular functions observed in cardiovascular disease states. The enhanced or unaltered levels of inhibitory G proteins (Giα) and mRNA have been reported in different models of hypertension, whereas Gsα levels are shown to be unaltered. The enhanced levels of Giα proteins precede the development of blood pressure and suggest that overexpression of Gi proteins may be one of the contributing factors for the pathogenesis of hypertension. The levels of vasoactive peptides including ET-1 and Ang II and growth factors are augmented in hypertension and contribute to the enhanced expression of Giα proteins in hypertension. In addition, oxidative stress due to enhanced levels of Ang II and ET-1 is enhanced in hypertension and may also be responsible for the enhanced expression of Giα proteins observed in hypertension. Furthermore, Ang II- and ET-1-induced transactivation of growth factor receptor through the activation of MAP kinase signaling is also shown to contribute to the augmented levels of Giα in hypertension. Thus, it appears that the enhanced levels of vasoactive peptides by increasing oxidative stress and transactivation growth factor receptors enhance MAP kinase activity that contribute to the enhanced expression of Giα proteins responsible for the pathogenesis of hypertension. In this review, we describe the role of vasoactive peptides and the signaling mechanisms responsible for the enhanced expression of Giα proteins in hypertension.

Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.

G protein signaling in the parasite Entamoeba histolytica

The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling-Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation.

Sitios de acción inhibitoria de la prolactina sobre la síntesis de estradiol inducida por la hormona folículo estimulante en las células de la granulosa / Sites of prolactin inhibition on gonadotropin-induced estrogen production in cultured rat granulosa cells

En este estudio se investigaron los sitios probables de la acción inhibitoria de prolactina (Prl) sobre la esteroidogénesis ovárica inducida por la hormona folículo estimulante (FSH). Para esta finalidad se estudió la capacidad de cultivos primarios de células de la granulosa de la rata de sintetizar estradiol y AMPc bajo la estimulación con FSH o de activadores de la vía dependiente de AMPc en presencia de Prl humana. La participación de otros sistemas de transducción de señal como los dependientes de PKC y proteínas Gi en los mecanismos de acción inhibitoria de la Prl fue también investigada utilizando inhibidores de estos sistemas como la calfostina C y la toxina pertusis. Los resultados demostraron la habilidad de la Prl de alterar la esteroidogénesis previa y posterior a la generación de AMPc, muy probablemente por mecanismos que involucran la activación de la subunidad catalítica de la adenilato ciclasa, así como a través de interactuar con sistemas de transducción de señal dependientes de PKC y proteínas sensibles a la toxina pertusis. Nuestros resultados sugieren un mecanismo de interacción entre receptores acoplados a proteínas G con aquéllos acoplados a cinasas de tirosinas mediado muy probablemente por vías de señalización dependientes de proteínas Gi.

We studied the sites of prolactin inhibition upon FSH induced ovarian steroidogenesis and the ability of prolactin (Prl) to inhibit the synthesis of estradiol and cAMP accumulation under the stimulation of FSH or cAMP dependent activators. The participation of other signal pathways such as PKC and Gi proteins on the inhibitory actions of Prl was also investigated using calfostine C andpertusis toxin as inhibitors. Results showed a dose dependent prolactin decrease in FSH-induced estradiol and cAMP production prior and after the generation of the cyclic nucleotide by a mechanism involving the catalytic subunit of adenyl cyclase and/or through activation of PKC or by the interaction with pertusin toxin sensitive G proteins. Our results suggest a mechanism by which G protein coupled receptors are linked with those coupled with tyrosine kinase through the involvement of a Gi protein mediated mechanism.

Signal transduction mechanism of antidepressant action / 中国临床药理学与治疗学

The mechanisms of antidepressants are still unclear. There a re two classical theories on monoamine neurotransmitter or on neurotransmitter rec eptors, but both of them can not fully explain the delayed therapeutic action of antidepressants. Recently, many researches have focused on the postreceptor int racellular signal transduction as the mechanism of antidepressant action. G protein is the molecular basis of antidepressants. Neurotransmitter receptors and G protein are the two sectors of their therapeut ic action. They will ultimately influence intracellular signal transduction and result in relative effects such as phosphoration, the induction of neurotrophic factors and neurogenesis. This mechanism suggests a reasonable explanation for t he clinical delaying of antidepressants and it will do great help for the develo pment of antidepressants. It makes the design of novel, safe and more efficaciou s antidepressants possible and provides significant information for the elucidat ion of biology of depression.

Receptor-specific Ca2+ signaling in polarized cells

No abstract available.

Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.

Increased expression of Galphaq protein in the heart of streptozotocin-induced diabetic rats

Heart disease is one of the major cause of death in diabetic patients, but the thogenesis of diabetic cardio-myopathy remains unclear. In this experiment, to sess the significance of G protein signaling pathways in the pathogenesis of abetic cardiomyopathy, we analyzed the expression of G proteins and the tivities of second messenger dependent protein kinases: cAMP-dependent protein nase (PKA), DAG-mediated protein kinase C (PKC), and calmodulin dependent otein kinase II (CaM kinase II) in the streptozotocin induced diabetic rat art. The expression of Galphaq was increased by slightly over 10% (P<0.05) in abetic rat heart, while Galphas, Galphai, and Gbeta remained unchanged. The A activity in the heart did not change significantly but increased by 27%<0.01) in the liver. Insulin treatment did not restore the increased activity the liver. Total PKC activity in the heart was increased by 56% (P<0.01), and sulin treatment did not restore such increase. The CaM kinase II activity in e heart remained at the same level but was slightly increased in the liver 4% increase, P<0.05). These findings of increased expression of Galphaq in the reptozotocin-diabetic rat heart that are reflected by the increased level of C activity and insensitivity to insulin demonstrate that alteration of Galphaq y underlie, at least partly, the cardiac dysfunction that is associated with abetes. Copyright 2000 Academic Press.

Inhibition of Neurite Outgrowth by Overexpression of Goa / 대한해부학회지

G proteins mediate signal transductions generated by neurotransmitters and hormones. Among G proteins, Go is found in a large quantity in brain, but its precise role in the nervous tissue is not fully understood. In addition, Go is one of the major proteins in growth cone membranes, which implies an important role of Go in the regulation of axon outgrowth. In this study, we attempted to determine the role of Go in axon outgrowth. We overexpressed the a subunit of Go (ao) in F11 neuroblatoma cells and examined the effect of ao on the neurite outgrowth. In F11 cells, dibutyryl cAMP increased neurite outgrowth remarkably upto 0.1 mM in a concentration dependent manner, but in a less degree at higher concentration. In the presence of 0.5 mM dibutyryl cAMP, the differentiation of F11 cells was almost saturated and the cells exhibited a typical neuronal morphology. Overexpression of ao caused a reduction of neurite outgrowth by 77.4% in length while increasing the number of neurites by 2.2 fold. The average neurite length was 38.9+/-12.5 mm in the ao-overexpressing F11 cells but 172.3+/-25.9 mm in the untransfected cells The total number of nurites per cell was 5.6+/-0.4 in the ao-overexpressing cells but 2.5 0.2 in the untransfected cells. This result suggests that Go may play an important role in growth cone collapse during neuronal cell differentiation.

Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain

C-terminal farnesyl cysteine carboxyl methylation has been known to be the last step in the post-translational modification processes of several important signal transduction proteins in eukaryotes including ras related GTP binding proteins and the gamma-subunit of heterotrimeric G proteins. Protein farnesyl cysteine carboxyl methyltransferase (PFCCMT; EC, 2.1.1.100) catalyzing the reaction is well characterized as being stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and suppressed by N-acetyl-S-farnesyl-L-cysteine (AFC). As an initial step to understand the physiological significance of the process, we attempted to purify the enzyme, which was partially purified 130-fold (specific activity, 143 pmol of methyl group transferred/min/mg of protein) with yield of 1.8% after purification by fast protein liquid chromatography (FPLC) on a Superdex 75 column. The enzyme was further purified with non denaturing polyacrylamide gel electrophoresis (ND-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of PFCCMT was determined to be about 30 kDa based on Superdex 75 FPLC as well as photoaffinity labelling with S-adenosyl-L-[methyl-3H] methionine ([methyl-3H]SAM). The partially purified enzyme (Superdex 75 eluate) was found to be characteristically affected by GTP gamma S, being activated about 40-fold in 2 mM, in contrast to ATP which did not show any effect on enzyme activity. Meanwhile, the enzyme was found to be markedly inhibited by AFC, reaching 0 activity in 2 mM. These observations strongly suggested that the partially purified enzyme was PFCCMT.

Retainment of membrane binding capacity of non-palmitoylated Gs alpha mutants expressed in COS-1 cells

Heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals into intracellular signals by coupling receptors and effectors. Because most of the G protein-coupled receptors are integral proteins, the G proteins need to have a membrane binding capacity to receive signals from the receptors. The alpha subunit of G protein binds tightly to the cytoplasmic face of the plasma membrane without any membrane spanning domain. Fatty acylation of G alpha with myristic acid or palmitic acid, in addition to the beta gamma subunits, plays an important role in anchoring the G alpha subunit. The reversible and dynamic palmitoylation of the alpha subunit of stimulatory G protein (Gs alpha) has been suggested as essential for its membrane attachment. However, in our previous experiments, Gs alpha deleted in the amino terminus containing palmitoylation site, retained its binding capacity when expressed in COS cells. Thus, to evaluate the role of palmitoylation in Gs alpha membrane binding, we constructed and expressed non-palmitoylated mutants of Gs alpha and analyzed their subcellular distributions in COS-1 cells. We found that non-palmitoylated mutants of Gs alpha, C3S- and G2A/C3S Gs alpha, retained their membrane binding capacities in COS-1 cells, demonstrating that palmitoylation is not essential for membrane binding of Gs alpha in COS-1 cells. We also found that the palmitoylation did not change significantly the distribution of Gs alpha in Triton X-114 partition. These results suggest that the palmitoylation of Gs alpha may produce different effects on membrane binding depending on cell types.

Regulation of cellular signals by G-proteins.

Extracellular signals are transduced across the cell by the cell surface receptors, with the aid of G-proteins, which act at a critical point of signal transduction and cellular regulation. Structurally, G-proteins are heterotrimeric consisting α, β and γ subunits but in functionally active state they dissociate into α subunit coupled to GTP and as βγ dimer. G-proteins can be broadly divided into two classes based on their sensitivity to pertussis toxin and cholera toxin. Existence of various forms of each of the subunit allows molecular diversity in the subunit species of G-proteins. These subunits interact with a wide range of receptors and effectors, facilitated by post translational modification of their subunits. Different types of G-proteins mediate several signalling events in different parts of the body. This review summarizes the features of (i) structural and functional heterogenity among different subunits of G-proteins, (ii) interaction of G-proteins and their subunits with effectors with specific cases of G-protein mediated signalling in olfaction, phototransduction in the retina, ras and ras related transduction and (iii) disease conditions associated with malfunctioning of G-proteins.

Expression of signal transducing G proteins in human melanoma cell lines

Some malignant melanoma cells regress spontaneously by terminal differentiation, and understanding the mechanisms of this spontaneous regression can contribute to the development of a new therapy not only for melanoma but also for other cancers. The signal transducing G protein is one component of the signaling pathways for the differentiation-inducing molecules such as alpha-melanocyte-stimulating hormone (alpha-MSH) and cAMP. To investigate the role of G proteins in the differentiation process, we analyzed the expression of various G proteins by quantitative Western blot and cAMP response in human malignant melanoma cell lines. SK-MEL-3 cells expressed the largest amount of stimulatory G protein alpha subunit (G(s) alpha) and the largest amount of inhibitory G protein alpha subunit (G(i) alpha) was expressed in Malme-3M cells among the 4 melanoma cell lines analyzed in this experiment. The SK-MEL-28 cells exhibited largest amount of alpha subunit of G(q) and the beta subunits. The cAMP formation by forskolin stimulation was largest in the Malme-3M. The amount of cAMP formation did not show any correlation with the expression of G(s) alpha nor that of G(i) alpha. The population doubling time was longest in Malme-3M cells. In this experiment, we found that the melanoma cells vary widely both in the expression of various G proteins and in cAMP production depending on the cell lines.

Endothelium-Dependent Vasodilation Responses in the Patients with Essential Hypertension

BACKGROUND: Patients with essential hypertension have abnormal endothelium-dependent vasodilation due to reduced release of nitric oxide(NO). But it is uncertain whether this defect is selective for some pathways of nitric oxide production or a more generalized abnormality of endothelial function. The purpose of this study was to determine whether this defect is due to an impairment at the specific intracellular signal-transduction pathway level or is a more generalized endothelial dysfunction METHOD: Forearm blood flow was measured in 10 patients with essential hypertension (mean blood pressure, 129+/-16.6mmHg ; aged 48+/-10 years old) and 10 control subjects(mean blood pressure, 99.7+/-6.6mmHg ; aged 41+/-10 years old) using strain-gauge plethysmography. The endothelium-dependent vasodilators were acetylcholine(7.5, 15, and 30ug/min), which uses a pertussis toxin-sensitive signal transduction pathway, and bradykinin(100, 200, and 400ng/min), which uses a pertussis toxin-insensitive signal transduction pathway to activate nitric oxide production. Sodium nitroprusside(0.8, 1.6, and 3.2ug/min) was used as an endothelium-independent vasodilator. All drugs were infused into the brachial artery and the order was determined randomly. RESULTS: The maximum flow in response to acetylcholine was markedly impaired in hypertensive patients compared with control subjects(5.29+/-1.86 vs 11.04+/-2.46ml/min/100ml forearm tissue, p<0.001). But the maximum forearm blood flow in response to bradykinin was similar in the two groups(11.96+/-3.57 vs 12.48+/-1.92ml/min/100ml forearm tissue, p=0.69) and that in response to sodium nitroprusside was also similar(10.63+/-3.74 vs 10.51+/-2.39ml/min/100ml forearm tissue, p=0.94). CONCLUSION: Patients with essential hypertension have impaired endothelium-dependent vasodilator response only to acetylcholine, while the response to bradykinin is preserved. This finding indicates that the impairment of endothelial vasodilator function is selective, and suggests the defect occurs at the level of the intracellular signal transduction pathway.

hsp 83 mutation is a dominant enhancer of lethality associated with absence of the non-protein coding hsrω locus in Drosophila melanogaster.

The hrsω or the 93D heat shock locus of Drosophila melanogaster, which does not code for any protein, has an important role in development since nullosomy of this locus in transheterozygotes for two overlapping deficiencies, viz., Df(3R)eGp4 (eGp4) and Df(3R)GC14 (GC14), is known to cause a high (~ 80%) mortality with the small number of escapee nullosomic flies being sterile, weak and surviving for only a few days. We now show that a majority of the hsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of the hsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when one hsp83 mutant allele (hsp83e4A) is introduced in eGp4/GC14 trans-heterozygotes, practically none of the hsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction between hsp83 and hsrω genes was further confirmed by examining the effect of the hsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that the hsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for the hsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development of Drosophila reveals novel aspects of their biological functions.

Vesicular transport with emphasis on exocytosis

The eukaryotic cell is compartmentalized by a series of vesicular organelles which constitute the endocytic and exocytic transport pathways. Each vesicular compartment has distinct sets of membrane proteins, structures and functions. Despite continuous vesicular transport, each vesicular compartment maintains its structure and function by use of retention and retrieval signal for its own resident proteins. Proteins in transit along the endocytic and exocytic pathway are transported without admixing with cytoplasmic constituents by successive steps of budding from the donor vesicles, formation of intermediate transport vesicles, transport, targeting to and fusion with acceptor vesicles. Specificity and fidelity of the vesicular transport are conferred by vesicular membrane proteins and small molecular weight GTP-binding proteins of the Rab subfamily. Proteins for export are packaged into specific vesicles for their final destinations. Insertion into and retrieval from the plasma membrane of transport proteins in response to cellular stimulus are a new paradigm of cellular regulatory mechanism. Secretion of neurotransmitters, hormones and enzymes by exocytosis involves a complex set of cytosolic proteins, G-proteins, proteins on the secretory granule membrane and plasma membrane. Much progress has been recently made in identifying proteins and factors involved in the exocytosis. But the molecular interactions among identified proteins and regulatory factors are unknown and remain to be elucidated. Finally our chemiosmotic hypothesis which involves the H+ electrochemical gradient across the secretory granule membrane generated by an ATP-dependent electrogenic H(+)-ATPase as the potential driving force for fusion and release of granule contents will be discussed.

Roles of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1 / 中国病理生理杂志

AIM: To study the roles and mechanisms of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1(ET-1). METHODS: (1) Neonatal rat cardiomyocyte hypertrophic response was assayed by measuring cell surface area and protein content; (2) ERKs activity was determined by Whatman Paper Filter method; (3) Intracellular free calcium concentration ([Ca 2+ ]i) was measured using Fura-2/AM as a fluorescent indicator. RESULTS: (1) ET-1 could increase total protein production, surface area, ERKs activity and [Ca 2+ ]i in cultured cardiomyocyte in dose-dependent manner at concentrations ranging from 10 -9 to 10 -7 mol/L. And this effect could be abolished by BQ123, an antagonist of ET A receptor, partly inhibited by PTX, but not by BQ788, an antagonist of ET B receptor.(2)The activation of ERKs and the increase of [Ca 2+ ]i induced by ET-1 were obviously inhibited by PD98059, a selective ERKs kinase inhibitor, and nifedipine, a calcium channel blocker, respectively. Both antagonists partially inhibited ET-1-stimulated cardiomyocyte hypertrophic response. (3) Staurosporine, a selective PKC inhibitor, could inhibit ET-1-stimulated cardiomyocyte hypertrophic response and increase of [Ca 2+ ]i, but not affect the activation of ERKs. CONCLUSION: Cardiomyocyte hypertrophic response induced by ET-1 is mediated by ET A receptor coupled to PTX-sensitive G-protein, which involves at least two signalling pathways: PKC-mediated increase of [Ca 2+ ]i , and PKC-independent activation of ERKs. [