Activation Of Second Messenger Systems Research Articles

Estrogen-IGF-1 interactions in neuroprotection: ischemic stroke as a case study.

The steroid hormone 17b-estradiol and the peptide hormone insulin-like growth factor (IGF)-1 independently exert neuroprotective actions in neurologic diseases such as stroke. Only a few studies have directly addressed the interaction between the two hormone systems, however, there is a large literature that indicates potentially greater interactions between the 17b-estradiol and IGF-1 systems. The present review focuses on key issues related to this interaction including IGF-1 and sex differences and common activation of second messenger systems. Using ischemic stroke as a case study, this review also focuses on independent and cooperative actions of estrogen and IGF-1 on neuroprotection, blood brain barrier integrity, angiogenesis, inflammation and post-stroke epilepsy. Finally, the review also focuses on the astrocyte, a key mediator of post stroke repair, as a local source of 17b-estradiol and IGF-1. This review thus highlights areas where significant new research is needed to clarify the interactions between these two neuroprotectants.

Novel G protein‐ and GPCR‐Regulated Oncogenic Signaling Circuitries

The family of G protein‐coupled receptors (GPCRs) constitutes the largest group of cell surface proteins involved in signal transduction. GPCRs participate in a wide variety of physiological and pathophysiological functions, and are the target of 50–60% of all current therapeutic agents. This large receptor family plays important roles in normal and tumor cell growth. For example, the Kaposi's sarcoma (KS) associated herpesvirus (KSHV), the infectious cause of KS, expresses an oncogenic constitutively active GPCR, vGPCR, thus providing a direct link between G protein‐linked receptors and viral‐associated malignancies. Recent large cancer sequencing initiatives have revealed an unexpected and surprisingly high incidence of GPCR mutations, ranging from 5% to nearly 30%, in some of the most prevalent human malignancies. Activating mutations in Gαs have been observed in multiple tumors, while genes for both Gαq family members, Gαq (GNAQ) and Gα11 (GNA11), have been recently identified in approximately 80% of uveal melanomas, where they represent the driving uveal melanoma oncogene. How Gαq and its coupled receptors transduce mitogenic signals is still unclear, due to the complexity of signaling events perturbed upon Gq activation. Using a synthetic biology approach and a genome‐wide RNAi screen in drosophila cells, we have found that a highly conserved guanine nucleotide exchange factor, Trio, is essential to activate Rho‐ and Rac‐regulated signaling pathways acting on JNK and p38, and to transduce proliferative signals from Gαq to the nucleus independently of PLC‐β. Evidence will be presented that while many biological responses elicited by Gq depend on the transient activation of second messenger system, Gq utilizes a hardwired protein‐protein interaction‐based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth. This knowledge may provide opportunities for the development of new G protein‐ and GPCR‐targeted treatment options in cancer.

A Genome-wide RNAi Screen Reveals a Trio-Regulated Rho GTPase Circuitry Transducing Mitogenic Signals Initiated by G Protein-Coupled Receptors

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-β. Indeed, whereas many biological responses elicited by Gq depend on the transientactivation of second-messenger systems, Gq utilizesa hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.

Neurosteroid migration to intracellular compartments reduces steroid concentration in the membrane and diminishes GABA‐A receptor potentiation

Neurosteroids are potent modulators of GABA-A receptors. We have examined the time course of development of potentiation of alpha1beta2gamma2L GABA-A receptors during coapplication of GABA and an endogenous neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP). The simultaneous application of 3alpha5alphaP with 5 microm GABA resulted in a biphasic rising phase of current with time constants of 50-60 ms for the rapid phase and 0.3-3 s for the slow phase. The properties of the rapid phase were similar at all steroid concentrations but the time constant of the slower phase became successively shorter as the steroid concentration was increased. Potentiation developed very rapidly (tau = 130 ms) when cells were preincubated with 300 nm 3alpha5alphaP before application of GABA + 3alpha5alphaP, and in outside-out patch recordings, suggesting that steroid diffusion to intracellular compartments competes with receptor potentiation by depleting the cell membrane of steroid. Very low steroid concentrations (3-5 nm) potentiated GABA responses but the effects took minutes to develop. Intracellular accumulation of a fluorescent steroid analogue followed a similar time course, suggesting that slow potentiation results from slow accumulation within plasma membrane rather than indirect effects, such as activation of second messenger systems. In cell-attached single-channel recordings, where 3alpha5alphaP is normally applied through the pipette solution, addition of steroid to the bath solution dramatically shifted the steroid potentiation concentration-effect curve to lower steroid concentrations. We propose that bath-supplied steroid compensates for the diffusion of pipette-supplied steroid out of the patch to the rest of the cell membrane and/or intracellular compartments. The findings suggest that previous studies overestimate the minimum concentration of steroid capable of potentiating GABA actions at GABA-A receptors. The results have implications for the physiological role of endogenous neurosteroids.

Assay of G protein-coupled receptor activation of G proteins in native cell membranes using [35S]GTP gamma S binding.

The interaction of a G protein-coupled receptor (GPCR) with the G protein is the first step in the transduction of receptor binding to the activation of second-messenger systems mediated by G proteins. Binding of the poorly hydroylzable GTP analog guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]-GTP gamma S) to the alpha subunit of a G protein has been used as a biochemical assay to measure the efficacy of a compound. The maximal percent stimulation of [35S]GTP gamma S binding, induced by an agonist, correlates well with the efficacy of an agonist in an in vivo system. The concentration of agonist necessary to achieve 50% of the maximal stimulation is indicative of the affinity of the agonist for the receptor, under the assay conditions. The [35S]GTP gamma S binding assay, which uses membranes from either myeloma cells or endogenous tissues, is a biochemical assay to determine the efficacies of agonists for receptors that are expressed endogenously. Novel compounds that have been shown to have high affinity in radioligand receptor-binding assays are screened in the [35S]GTP gamma S binding assay to determine if they are agonists, antagonists, or partial agonists at a particular receptor.

Haemodynamic action of B‐type natriuretic peptide substantially outlasts its plasma half‐life in conscious dogs

1. The objective of the present study was to determine the plasma half-life of B-type natriuretic peptide (BNP) in conscious dogs after intravenous administration and to compare this with its haemodynamic effects. In six chronically instrumented dogs, plasma BNP concentrations were measured under basal conditions, during a constant infusion of canine BNP-32 (10 pmol/kg per min; 25 min) to steady state and at nominated time points up to 75 min after stopping the infusion. Concomitant, continuous measurements of mean arterial blood pressure (MAP), heart rate (HR), central venous pressure (CVP) and mesenteric blood flow (MBF) were obtained. 2. Baseline plasma BNP levels were 15.0 +/- 2.3 fmol/mL and rose approximately 10-fold to 159 +/- 23 fmol/mL after 20-25 min BNP infusion. When the infusion was turned off, plasma BNP levels declined in a biphasic manner, with an initial half-life of 1.57 +/- 0.14 min and a terminal half-life of 301 +/- 85 min. The metabolic clearance rate of BNP was 2.29 +/- 0.34 L/min. 3. The infusion of BNP reduced MAP (approximately 10%), CVP (approximately 65%) and MBF (approximately 25%), whereas haematocrit (approximately 4%) and mesenteric vascular resistance (MVR) increased (approximately 40%; all P < 0.05). Plasma BNP levels returned to baseline by 20 min after BNP infusion had been stopped, whereas none of the haemodynamic variables returned to normal by this time. Mean arterial pressure returned to resting levels within 10-15 min after plasma BNP returned to normal. However, CVP, haematocrit and MBF remained substantially below baseline values for more than 20 min after circulating BNP levels had returned to pre-infusion levels. Of these, only mesenteric vascular changes were returned to baseline within 60 min of plasma BNP levels normalizing. 4. These results demonstrate that the removal of BNP from the canine circulation is rapid, similar to observations made regarding the metabolism of circulating atrial natriuretic peptide in dogs. The half-life of BNP in dogs was shorter than that in rats, sheep or humans. However, the haemodynamic actions of BNP substantially outlasted its plasma half-life. Whether this disparity in plasma level and haemodynamic activity of BNP reflects long-lasting activation of second messenger systems or slow recovery from the hydraulic changes at the capillary level, reflected in the haematocrit and CVP, remains to be answered.

Endothelium-dependent effects of estrogen on vasomotor tone: Consequences of nongenomic actions

Clinical observational and experimental studies indicate that estrogen has acute and prolonged effects on vascular function. Acute effects not requiring gene transcription or nongenomic responses include changes in intracellular calcium and activation of second messenger systems including release of endothelium-derived nitric oxide. However, interactions between rapid effects of estrogen and modulation of subsequent responses of endothelial cells are lacking. Experiments were designed to determine effects of estrogen on subsequent responses of endothelial cells to vasoactive stimuli. Human umbilical vein endothelial cells were cultured on coverslips and changes in intracellular calcium concentrations measured to 17β-estradiol and histamine. Estradiol alone did not increase intracellular calcium or modulate the peak calcium response to histamine. However, the decay of the peak calcium response to histamine was more rapid in the presence, compared to the absence, of estradiol. These results provide proof of principal that estradiol modulates responses of endothelial cells to vasoactive stimuli through changes in regulation of intracellular calcium.

15] Antisense approach for study of cell adhesion molecules in central nervous system

All cell types studied to date use multiple molecular mechanisms to adhere to other cells and to the extracellular matrix. Thus, the specificity of cell–cell and cell–matrix adhesion must result from the integration of a number of different adhesion systems, some of which are associated with specialized cell junctions while others are not. The adhesive potential of these glycoproteins determines their general designation as cell adhesion molecules (CAMs) and substrate adhesion molecules (SAMs). However, these operational designations do not always accurately reflect their biological functions. An increasing body of evidence suggests that the biological activities of several CAMs may be accounted for by a direct activation of second-messenger systems, probably by their participation in signal transduction. Although their conventional name, CAMs, does not accurately describe their complex biological functions, a more appropriate name such as “recognition molecules” was never fully accepted by the research community. Hence, the term “CAM” is generally used.

Molecular Neurobiology for Practicing Psychiatrists, Part 3

One of the most important advances in molecular neurobiology of relevance to the practicing psychiatrist is how an intracellular second messenger can "turn on" genes by activating first a protein kinase enzyme and then a transcription factor. Failure to turn on the right genes may lead to psychiatric illnesses. Causing the appropriate genes to turn on may be the therapeutic mechanism of action of many current and future psychotropic drugs.

CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

Dual 1,25-dihydroxyvitamin D3 signal response pathways in osteoblasts: Cross-talk between genomic and membrane-initiated pathways

Osteoblasts are key regulatory cells in the control of systemic calcium ion (Ca2+) homeostasis. They, along with cells in the kidney and intestine, function as an integral part of the vitamin D endocrine system. The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), interacts with osteoblasts at several levels to modulate their phenotype and function. The interactions involve distinct receptor systems that operate on unique time scales. Rapid nongenomic actions (milliseconds to minutes), mediated through membrane receptor systems, do not require protein synthesis and include activation of voltage-sensitive Ca2+ channels, induction of phospholipid and sphingolipid turnover, elevation of intracellular Ca2+ concentrations, priming of parathyroid hormone (PTH)-sensitive ion channels, and activation of second messenger systems. In the longer term (many hours to days), interactions mediated through binding of 1,25(OH)2D3 to nuclear receptors, present in mature osteoblasts, modulate transcription of target genes. Target genes for 1,25(OH)2D3 including those encoding for the bone matrix proteins osteopontin (OPN) and osteocalcin (OCN), possess vitamin D response elements (VDRE) upstream of the transcriptional start site. In addition, it is now clear that a number of intermediate effects (1 to 3 hours) also occur. These effects require longer times than the aforementioned rapid effects, but precede the long-term consequences of activation of nuclear receptors. These include alterations in the phosphorylation state of various proteins secreted by osteoblasts, including OPN, attributable to activation/inactivation of various intracellular protein kinases and phosphatases. Intermediate effects are likely to involve both membrane-initiated rapid actions and transcriptional effects on early genes that do not require the nuclear receptor for 1,25(OH)2D3. These intermediate effects, therefore, represent ideal targets for the study of the interaction or “cross-talk” between rapid membrane-initiated, nongenomic effects and nuclear receptor-mediated, genomic effects of 1,25(OH)2D3 on osteoblasts. Intermediate effects also invite the study of rapid, non-nuclear receptor-mediated genomic effects of 1,25(OH)2D3 on osteoblasts. Complete understanding of the mechanisms by which 1,25(OH)2D3 exerts its pleiotropic effects on osteoblasts and other target cells, contributing to control of Ca2+ homeostasis, requires integration of the concepts learned from studies of both rapid and long-term pathways. (Am J Kidney Dis 1998 Apr;31(4):729-42)

Characterization and partial purification of a keratinocyte-derived growth factor with wound-healing properties.

We have previously described the mitogenic and wound-healing properties of keratinocyte-conditioned medium (KCM). In this study we investigated the effect of KCM on the activation of second messenger systems and the expression of proto-oncogene in cultured human skin fibroblasts. We also present a partial purification of the factor responsible for the mitogenic and wound-healing effects of KCM. KCM was shown to increase the expression of the proto-oncogenes c-fos, c-myc and c-jun. The effect of KCM on three second messenger systems was investigated. The extracellular release of choline metabolites was increased by 40 per cent when cells were stimulated with KCM whereas the formation of cAMP and hydrolysis of phosphatidyl inositol (PI) was unaffected. KCM was purified by ion exchange chromatography and filtration. The biologically active fraction was eluted from an SP column and retained its activity after filtration through a 3-kDa filter. The fraction was inactivated by heat and acid, indicative of a peptide origin. Furthermore, the active fraction was shown to increase the extracellular release of choline metabolites and to stimulate re-epithelialization in wounds in human skin in vitro comparable to KCM. The study indicates that human keratinocytes produce a < 3 kDa peptide which may be partly responsible for the growth stimulatory and wound-healing properties of KCM.

Role of the Ca2+-Sensing Receptor in Divalent Mineral Ion Homeostasis

The divalent mineral cations Ca2+ and Mg2+ play many and diverse roles both in the function of cells and in extracellular processes. The metabolism of these cations is a complex process involving the coordinated function of several organ systems and endocrine glands. A recently cloned G-protein-coupled receptor responds to extracellular calcium concentration (Ca2+0-sensing receptor, CaSR) and mediates several of the known effects of Ca2+0 on parathyroid and renal function. The CaSR, which is also expressed in a number of other tissues including thyroidal C-cells, brain and gastrointestinal tract, may function as a Ca2+0 sensor in these tissues as well. Thus, Ca2+0 is a first messenger (or hormone) which, via CaSR-mediated activation of second messenger systems (e.g. phospholipases C and A2, cyclic AMP) leads to altered function of these cells. Several mutations in the human CaSR gene have been identified and shown to cause three inherited diseases of calcium homeostasis, clearly implicating the CaSR as an important component of the homeostatic mechanism for divalent mineral ions. Ca2+ and Mg2+ losses from the body are regulated by altering the urinary excretion of these divalent cations. The localization of the CaSR transcripts and protein in the kidney not only provides a basis for a direct Ca2+0 (or Mg2+0)-mediated regulation of Ca2+ (and Mg2+) excretion but also suggests a functional link between divalent mineral and water metabolism. In the kidney, the thick ascending limb of Henle (TAL) plays crucial roles in regulating both divalent mineral reabsorption and urine concentration. Recent studies have suggested models whereby extracellular Ca2+, via the CaSR expressed in the TAL as well as in the collecting duct system, modulates both Ca2+ 0 and Mg2+ 0 as well as water reabsorbtion. When taken together, these studies suggest that the CaSR not only provides the primary mechanism for Ca2+ 0-mediated regulation of parathyroid hormone secretion from parathyroid glands but also for direct modulation of renal divalent mineral excretion and urinary concentrating ability. These latter functions may furnish a mechanism for integrating and balancing water and divalent cation losses that minimizes the risk of urinary tract stone formation. This mechanism can explain hypercalcemia-mediated polyuria (diabetes insipidus).

Role of interneurons in defensive withdrawal reflexes in Aplysia.

Introduction Much attention has been focused on the contr ibution of sensory neurons and their synaptic connections to defensive reflexes in Aplysia. The two best understood withdrawal reflexes in terms of their underlying circuitry and their roles in learning and memory, are the siphon-elicited siphon-gill withdrawal reflex and the tail-elicited tail-siphon withdrawal reflex (Fig. 1; for review, see Kandel and Schwartz 1982; Carew and Sahley 1986; Hawkins et al. 1986; Byrne 1987; Byrne et al. 1991; Frost and Kandel 1995; Byrne and Kandel 1996). Both reflex responses have been used extensively to analyze the neural and molecular mechanisms of sensitization, a simple form of nonassociative learning in which an unexpected (often noxious) stimulus enhances the response to a subsequent test stimulus (for review, see Byrne et al. 1993; Hawkins et al. 1993). Another form of nonassociative learning, habituation, has also been the subject of study, although to a lesser extent. The duration of both forms of learning can be brief (minutes) or long (days), depending on the training protocol. Much of the work on the mechanisms underlying sensitization in these two reflexes has focused on training-related modifications of the central sensory neurons. In particular, sensitizing stimuli act to enhance both sensory neuron excitability and transmitter release (for review, see Byrne et al. 1993; Hawkins et al. 1993). These changes occur via the activation of second messenger systems in a timeand state-dependent manner (Baxter and Byrne 1990; Braha et al. 1990; Ghirardi et al. 1992; Pieroni and Byrne 1992; Sugita et al. 1992, 1994; Byrne and Kandel 1996). Several of these storage processes operate in parallel to maintain both the shortand long-term components of the memory for sensitization (Frost et al. 1985; Greenberg et al. 1987; Schwartz and Greenberg 1987; Scholz and Byrne 1988; Bailey et al. 1992; Kennedy et al. 1992; Byrne et al. 1993; Alberini et al. 1994; O'Leary et al. 1995). More recent studies have begun to explore the role of circuit interneurons in both the mediation and modulation of these withdrawal responses. This review will address several questions about the roles played by the interneurons in these circuits. What do the interneurons contr ibute to the mediation of these reflexes? Does their contr ibution differ qualitatively from that made by the monosynaptic sensory neuron to motor neuron connections? Do the interneurons themselves serve as sites of memory storage and, if so, do they use the same storage mechanisms as those utilized by the sensory neurons? What role do interneurons play in learning-related modulation of the circuits? The findings reviewed here indicate that interneurons make important and distinctive contributions to the mediation of withdrawal reflexes in Aplysia. Furthermore, some of the interneurons serve as sites of information storage in habituation and sensitization, storing different 1Corresponding author, information, and utilizing different types of plasticity than the sensory

Synthesis of models for excitable membranes, synaptic transmission and neuromodulation using a common kinetic formalism.

Markov kinetic models were used to synthesize a complete description of synaptic transmission, including opening of voltage-dependent channels in the presynaptic terminal, release of neurotransmitter, gating of postsynaptic receptors, and activation of second-messenger systems. These kinetic schemes provide a more general framework for modeling ion channels than the Hodgkin-Huxley formalism, supporting a continuous spectrum of descriptions ranging from the very simple and computationally efficient to the highly complex and biophysically precise. Examples are given of simple kinetic schemes based on fits to experimental data that capture the essential properties of voltage-gated, synaptic and neuromodulatory currents. The Markov formalism allows the dynamics of ionic currents to be considered naturally in the larger context of biochemical signal transduction. This framework can facilitate the integration of a wide range of experimental data and promote consistent theoretical analysis of neural mechanisms from molecular interactions to network computations.

The effect of trans-ACPD, a metabotropic excitatory amino acid receptor agonist, on the responses of primate spinothalamic tract neurons

The responses of primate spinothalamic tract (STT) neurons to innocuous and noxious mechanical stimuli applied to the skin can be enhanced for more than an hour following prolonged noxious stimulation. This increased responsiveness is thought to reflect sensitization of dorsal horn neurons and may help account for secondary hyperalgesia and mechanical allodynia. The proposal that central sensitization is due to the activation of second messenger systems was tested in this study by examining the effect of trans-ACPD ( trans- d,l-1-amino-1, 3-cyclopentanedicarboxylic acid), an agonist of metabotropic excitatory amino acid (EAA) receptors, introduced into the dorsal horn by microdialysis. A low dose of trans-ACPD resulted in an increase in the responses of STT cells to an innocuous mechanical stimulus (BRUSH), but no increase in the responses to noxious mechanical and thermal stimuli or in the excitation produced by iontophoretically applied EAAs. A high dose of trans-ACPD caused a transient increase in background activity, but no change in the responsiveness of spinothalamic cells to any of the test stimuli. It is concluded that low doses of trans-ACPD can selectively enhance transmission through interneuronal pathways mediating tactile inputs to spinothalamic cells.

Activation of second messenger systems can enhance or inhibit the secretion of the amyloid precursor protein (APP) : M.R. Emmerling, P.D. Doyle, C.J. Moore, R.T. Carroll, and R.E. Davis, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., 2800 Plymouth Road, Ann Arbor, MI 48106 USA

Patients suffering of Alzheimer's disease (AD) are characterized by a low transthyretin (TTR) level in the brain. The effect of pH and TTR concentration in the medium on the β-amyloid protein (Aβ)/heparan sulfate proteoglycan (HSPG) association mechanism were studied using a biochromatographic approach. For this purpose, HSPG was immobilized via amino groups onto the amino propyl silica pre-packed column, activated with glutaraldehyde, by using the Schiff base method. Using an equilibrium perturbation method, it was clearly shown that Aβ can be bound with HSPG. This approach allowed the determination of the thermodynamic data of this binding mechanism. The role of the pH was also analyzed. Results from enthalpy–entropy compensation and the plot of the number of protons exchanged versus pH showed that the binding mechanism was dependent on pH with a critical value at pH = 6.5. This value agreed with a histidine protonation as an imidazolium cation. Moreover, the corresponding thermodynamical data showed that at pH > 6.5, van der Waals and hydrogen bonds due to aromatic amino acids as tyrosine or phenylalanine present in the N-terminal (NT) part governed the Aβ/HSPG association. Aβ remained in its physiological structure in a random coil form (i.e. the non-amyloidogenic structure) because van der Waals interactions and hydrogen bonds were preponderant. At acidic pH (pH < 6.5), ionic and hydrophobic interactions, created by histidine protonation and hydrophobic amino acids, appeared in the Aβ/HSPG binding. These hydrophobic and ionic interactions led to the conversion of the random coil form of Aβ into a β-sheet structure which was the amyloidogenic folding. When TTR was incubated with Aβ, the Aβ/HSPG association mechanism was enthalpy driven at all pH values. The affinity of Aβ for HSPG decreased when TTR concentration increased due to the complexation of Aβ with TTR. Also, the decrease of the peak area with the increase of TTR concentration demonstrated that this Aβ/TTR association led to the cleavage of Aβ full length to a smaller fragment. For acidic pH (pH < 6.5), it was shown that the importance of the hydrophobic and ionic interactions decreased when TTR concentration increased. This result confirmed that Aβ was cleaved by TTR in a part containing only the NT part. Our results demonstrated clearly that TTR reversed the effect of acidic pH and thus played a protective role in AD.

Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells.

IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set.

Influence of internal calcium stores on calcium-activated membrane currents in smooth muscle.

Agonist-induced activation of second messenger systems that increase inositol 1,4,5-trisphosphate concentration plays an important role in the mobilisation of stored Ca2+ in smooth muscle. Release of Ca2+ which occurs spontaneously or by agonist stimulation causes activation of Ca(2+)-sensitive currents. Experiments using selective modulators of the Ca2+ uptake and release mechanisms of the sarcoplasmic reticulum provide evidence that depletion of the internal Ca2+ store triggers an influx of Ca2+ from the extracellular space. Recent data suggest that pool depletion causes the opening of non-selective cation channels which are possibly similar to those previously designated 'receptor-operated Ca2+ channels'. Ca2+ entry through these channels not only refills the internal Ca2+ pool, but also modulates membrane excitability by stimulating Ca(2+)-activated currents.

Retinoblastoma protein dephosphorylation induced by D-erythro-sphingosine.

The retinoblastoma gene product (Rb), a nuclear phosphoprotein, functions as a tumor suppressor that is inactivated in retinoblastoma and other malignancies. The hypophosphorylated forms of Rb are observed in the G0/G1 phase of the cell cycle, whereas the hyperphosphorylated forms predominate in S and G2/M phases, suggesting that phosphorylation/dephosphorylation of Rb may regulate progression through the growth cycle. However, little is known about the intracellular signals that regulate phosphorylation/dephosphorylation of Rb. We show that D-erythro-sphingosine potently induces early dephosphorylation of Rb. Initial dephosphorylation was observed as early as 1 h after treatment of hematopoietic cells with sphingosine, whereas complete shift to the dephosphorylated form was seen 4 h after treatment. These effects occurred at concentrations of sphingosine as low as 100-500 nM, with maximal effects observed at 1-2.5 microM. These effects were specific to sphingosine, inasmuch as other lipids, amphiphiles, and long chain amino bases, as well as structural analogs of sphingosine, failed to induce dephosphorylation of Rb. Also, activation of second messenger systems including protein kinase C, cAMP-dependent kinases, and calcium ionophores, as well as inhibition of serine/threonine protein phosphatases, failed to induce dephosphorylation of Rb. Induction of Rb dephosphorylation by sphingosine preceded inhibition of growth and a specific arrest in the G0/G1 phase of the cell cycle. These studies, for the first time, identify an intracellular activator of Rb.