Messenger Cascades Research Articles

P.5.035 - Characterization of [ 3H]GR 125,743 as a radioligand at 5-HT 1Dreceptors in guinea pig brain: A comparison with [ 3H]5CT

Venlafaxine, a dual amine reuptake inhibitor, was utilized to delineate the role of the individual aminergic components of the `serotonin/noradrenaline link' in modifying receptor-linked second messenger cascades. Venlafaxine (20 mg/kg i.p. bid for 10 days) failed to alter in normal animals either the density of β adrenoceptors or the response of the β adrenoceptor-coupled adenylate cyclase system to noradrenaline but significantly decreased the cyclic AMP response to noradrenaline in the brain of rats with selective depletion of brain serotonin by p-chlorophenylalanine. The studies provide evidence for a cross-talk between noradrenergic and serotonergic receptor cascades at the level of mechanisms involved in the desensitization of the β adrenoceptor-coupled adenylate cyclase system.

Relationship between protein kinase C and adenylyl cyclase activity in the regulation of growth hormone secretion by human pituitary somatotrophinomas.

To determine the potential role of protein kinase C (PKC) and its relationship to adenylyl cyclase activity in controlling growth hormone (GH) secretion by human pituitary somatotrophinomas. Twenty-eight somatotrophinomas were placed into cell culture, and the in vitro effects of the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) and the PKC inhibitor staurosporine on basal and GH-releasing hormone (GHRH)-stimulated GH secretion were examined. In addition, the influence of chronic exposure of cultured somatotrophinoma cells to TPA on the rate of inositol 1,4,5-trisphosphate production was determined. Each tumor was assessed for the presence of gsp oncogenes, and thus constitutive adenylyl cyclase activity, by direct sequence analysis of polymerase chain reaction-generated deoxyribonucleic acid. GH secretory responses of tumors with and without these oncogenes were compared. TPA consistently stimulated GH secretion by cultured somatotrophinoma cells. There was no difference in response between somatotrophinomas with and without gsp oncogenes, and the effects did not correlate with the variable stimulation exerted by GHRH. Tumors in which GHRH had no significant effect nevertheless responded to TPA. In combination, TPA and GHRH exerted additive stimulation. TPA treatment of cultured somatotrophinoma cells eventually resulted in suppression of inositol 1,4,5-trisphosphate production, probably reflecting down-regulation of membrane phosphatidylinositol hydrolysis, a second messenger system that also generates the endogenous PKC activator diacylglycerol. GHRH had no effect on phosphatidylinositol hydrolysis. In contrast to the effects of TPA, the PKC inhibitor staurosporine tended to reduce GH secretion, although this effect was not observed in all tumors examined. As with TPA, the effects of staurosporine did not correlate with presence or absence of gsp oncogenes. Furthermore, staurosporine did not reduce the stimulatory effects exerted by GHRH on GH secretion. These results demonstrate a role for the phosphatidylinositol-PKC second messenger cascade in controlling GH secretion by human pituitary somatotrophinomas. The results also show that the system operates relatively independent of intracellular adenylyl cyclase and, thus, protein kinase A.

D2 dopamine receptor-mediated inhibition of a hyperpolarization-activated current in rod photoreceptors.

1. Using the whole cell patch clamp method, we investigated the effect of dopamine on a hyperpolarization-activated current (Ih) in the inner segments of rod photoreceptors of the Xenopus retina. 2. Ih was elicited by hyperpolarizing voltage steps to -120 mV from a holding potential of -40 mV. Dopamine reversibly reduced Ih in a dose-dependent manner. Dopamine-mediated inhibition of Ih was blocked by the D2 dopamine antagonist sulpiride. 3. The D2 dopamine agonist quinpirole (0.1-20 microM) inhibited Ih whereas the D1 agonist SKF-38393 (100 microM) had no effect on Ih. Quinpirole-induced inhibition of Ih was blocked by sulpiride, but not by the D4 antagonist, clozapine. The D3 agonists (+/-)-7-hydroxy-2-dipropylaminotetralin hydrochloride and trans-7-hydroxy-2[N-propyl-N-(3'-iodo-2'-propenyl)amino]-tetralin maleate were, respectively, 5 and 100 times less effective than quinpirole in inhibiting Ih. 4. Quinpirole failed to reduce Ih when the internal solution contained GDP beta S (500 microM). Internal application GTP gamma S (300 microM) progressively and irreversibly reduced Ih and blocked a further reduction by quinpirole, indicating that the inhibition of Ih by quinpirole involves a G protein. 5. The inhibition of Ih by quinpirole was not affected by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP) or by the protein kinase inhibitor H-7, indicating that a cAMP-mediated second messenger cascade does not participate in the dopamine-mediated inhibition. 6. Ih was not altered when the patch pipette contained a nominally Ca(2+)-free internal solution, but the inhibition of Ih by quinpirole was abolished, suggesting an involvement of Ca(2+) in the quinpirole-induced effect. 7. We conclude that a D2 dopamine receptor modulates Ih through the activation of a G protein and that intracellular Ca2+, but not cAMP, plays a key role in this process. 8. The reduction of Ih by dopamine may reduce the ability of rods to signal time-modulated light stimuli.

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: Regulation by calcium, protein kinases, and protein phosphatases

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actincrosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5–10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin. © 1996 Wiley-Liss, Inc.

Bone resorption and mRNA expression of IL-6 and IL-6 receptor in patients with renal osteodystrophy

The cytokine interleukin-6 (IL-6) is a major cell regulatory factor that may play an important role in the bone remodeling of patients with renal failure. IL-6 exerts its action by binding to its receptor (IL-6R), which leads to transduction of a second messenger cascade within cells. In vitro as well as in vivo data point to IL-6 as an autocrine/paracrine factor in bone osteoclasts. Recently, bone cells from patients with Paget's disease were found to express IL-6 and IL-6R mRNA transcripts. However, in patients with renal bone disease, there is currently no in vivo evidence that osteoclasts have the capability to express mRNA for IL-6 and IL-6R. To investigate the potential expression of IL-6 and IL-6R in bone and its relationship to bone cell activity, iliac crest bone biopsies were performed in patients on chronic maintenance dialysis. Messenger RNA expression of IL-6 and IL-6R was studied using in situ hybridization histochemistry, and parameters of bone turnover were determined by bone histomorphometry. In the samples studied, mRNA expression of IL-6 and IL-6R was found in osteoclasts and bone marrow cells. Furthermore, we report the novel finding of increased IL-6R mRNA expression in osteoclasts engaged in increased bone resorption. The results of the present study suggest that the cytokine IL-6 is intricately involved in osteoclastic bone resorption and that expression of its receptor, IL-6R, in osteoclasts may parallel osteoclastic bone resorbing activity.

Receptor signalling and the regulation of endocytic membrane transport

Vesicle-mediated membrane traffic has long been considered to be a constitutive process that is not burdened by layers of regulation. This contrasts with transmembrane signalling systems at the plasma membrane which relay information (i.e. extracellular stimuli) from the cell surface to the cytoplasm via a myriad of different protein—protein interactions and second messenger cascades. An accumulation of recent evidence, however, now suggests that signal-transduction pathways also play a critical role in the regulation of protein and membrane trafficking. In particular, the analysis of the signalling pathways initiated by receptor tyrosine kinases at the plasma membrane has yielded new insights into the molecular mechanisms of endocytosis. In addition, recent evidence has suggested potential new roles for two previously characterized vesicle coat proteins in a membrane traffic route that is regulated via cell surface receptor signalling.

Discussion

Learning is the modification of behaviour by experience, and memory is the retention of such modifications. Since learning events might be of short duration, selective neuronal mechanisms must exist to translate transient activity into long-lasting memory. Because metabotropic glutamate (mGlu) receptors are coupled to various second messenger cascades they are ideal candidates for such translations. Their involvement in synaptic plasticity has been demonstrated recently, an important finding given that changes in synaptic efficacy are widely believed to be the physical substrate for information storage. Behavioural investigations using selective drugs have demonstrated that memory formation, especially of hippocampus-dependent tasks, is blocked by pre-training treatment with both mGlu-receptor agonists and antagonists. In contrast, agonists administered post-training might amplify memory formation. The hypothesis put forward here suggests that the primary function of mGlu receptors is to set the signal-to-noise ratio and thereby filter out unimportant or amplify important information.

Bombesin receptors in the brain.

We have shown that in the central nervous system BN receptors are closely associated with 5-HT systems. On a subpopulation of dorsal raphe neurons, NMB receptors are able to depolarize cells by reducing gK+. In one of the target regions of the dorsal raphe 5-HT neurons, the SCN, we have also shown that neurons are excited by BN-related peptides. In the SCN, the GRP receptors excite neurons by two different mechanisms: closure of gK+ and opening of an unidentified cation conductance. Expression of human BN receptors from the brain in CHO cells or Xenopus oocytes shows a very similar pharmacological profile to that seen in the rat brain slice preparations. In the CHO cell line, following BN receptor activation, a major second-messenger path involves hydrolysis of PIP2 by phospholipases to yield IP3, which releases Ca2+ from intracellular stores. In the oocyte expression system, a similar second messenger pathway is clearly apparent, and Ca2+-sensitive gCl- represents the last phase in a cascade of events. The final phase of the mechanism of action in the artificial systems does not involve gK+, suggesting a different second messenger cascade to that in neurons. However, the involvement of phospholipases and their phospholipid products have not been excluded in neurons.

Effects of ultraviolet light in vascular cells in vitro and in intact atherosclerotic explants: potential role of apoptosis in vascular biology

Complex cell-to-cell interactions are known to participate during vascular injury and remodeling, resulting in smooth muscle cell proliferation. Mechanical interventions have yielded little benefit in limiting this process and several site-specific genetic therapies are not yet clinically available. The aim of this study was to delineate the effect of very short wavelength ultraviolet (UVC) light therapy on the viability of macrophage and smooth muscle cells. Vascular cells were both treated in vitro and in intact explanted atherosclerotic aortic segments ex vivo with UVC light. Brief exposure to short wavelength UVC light in the absence of photosensitizers elicited a differential temporal and functional response among treated cells. However, dramatic reduction in both cellular viability and proliferative capacity with eventual cell demise was observed in all UVC-treated cells. Flow cytometry and immunohistochemical analyses revealed the presence of extensive DNA fragmentation, suggestive of apoptosis as a predominant pathway of cell death in these cells exposed to UVC light. We hypothesize that selective induction of apoptosis, in contrast to necrosis, with UVC light may represent a beneficial approach to interdict the complex biologic cascade of messengers that participate in the restenotic response to vascular injury.

Physiology and biochemistry of Drosophila learning mutants.

Single gene mutants of Drosophila that are defective in learning/memory processes have increased substantially our understanding of the physiology, biochemistry, and anatomy underlying conditioned behaviors. Drosophila learning mutants can be separated into two general classes, those with structural defects in the brain and those without (conditioning mutants) any obvious brain alterations. From studies of brain structural mutants, two neuroanatomic areas have merged as important for normal conditioned behavior: the mushroom bodies and the central complex. Biochemical and molecular genetic studies of the conditioning mutants have implicated numerous types of molecules in learning, but the adenosine 3',5'-cyclic monophosphate (cAMP) second messenger pathway has emerged as especially important. Five different genes in this pathway, amnesiac (a product similar to adenylate cyclase activating peptides), dunce (cAMP phosophodiesterase), rutabaga (adenylyl cyclase), DCO (protein kinase A), and dCREB2 (cAMP-response element binding protein), have proven important for normal learning. The products of many of these learning mutants are enriched in mushroom bodies, which highlight the importance of mushroom bodies for normal learning and the cAMP second messenger cascade for the physiology of mushroom body cells in their role(s) underlying learning. Physiological studies of the mutants have demonstrated that plastic properties of synaptic transmission, including facilitation and posttetanic potentiation, are abnormal in the mutants. An appendix describing the currently used paradigms to test Drosophila behavior is included.

Activation of metabotropic glutamate receptors induce differential effects on synaptic transmission in the dentate gyrus and CA1 of the hippocampus in the anaesthetized rat

Activation of ACPD-sensitive metabotropic receptors induced differential effects on synaptic transmission and the induction of LTP in CA1 and the dentate gyrus of the hippocampus i.c.v. injections of (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S,3 R)-ACPD] induced enduring potentiation of the fEPSP in CA1, which occluded tetanically induced LTP. In contrast, ACPD induced a dose-dependent biphasic effect on the fEPSP in the dentate gyrus, consisting of an initial short lasting potentiation, followed by enduring depression of the response, and blockade of LTP. These two effects are likely to be mediated by two different classes of the receptor as in the dentate gyrus the selective class I agonist, ( RS)-3,5-dihydroxyphenylglycine (DHPG) induced sustained potentiation of the fEPSP, whereas the mixed mGluR2 agonist-mGluR1 antagonist, ( S)-4-carboxy-3-hydrophenylglycine(( S)-4 C3H- PG) induced only depression. Increasing the concentration of calcium directly in the dentate gyrus prior to, and in conjunction with, injections of ACPD induced sustained potentiation rather than depression. The differential effects indicate that the second messenger cascades the subtypes of receptors are linked with, mediate different forms of synaptic plasticity within the hippocampus and have important implications for their role in learning.

NaF potentiates a K(+)-selective ion channel in G292 osteoblastic cells.

Clinical studies have established that NaF increases mineral content in bone, although the cellular mechanisms underlying its osteoinductive effects remain unclear. Because metabolic effects of fluoride have been linked to ion flux and alterations in membrane potential, we used patch-clamp recording techniques to examine the electrophysiological response of osteoblastic cells to NaF. In these experiments, we show that NaF increased the amplitude and P(open) of a 73 pS potassium-selective ion channel. The effect of NaF depended on extracellular Ca2+ and could be blocked by a combination of calcium-channel blocking agents, suggesting that potentiation of channel activity was dependent on external calcium. Because all patches were in the cell-attached configuration, the effect of NaF was presumable indirect. Although the underlying cellular mechanisms remain unclear, our findings suggest that activity of calcium and/or potassium-selective channels via second messenger cascades may mediate many of the early events involved in the response of bone cells to inorganic fluoride.

Review: a role for the FGF receptor in the axonal growth response stimulated by cell adhesion molecules?

Cell adhesion molecules (CAMs) have been shown to stimulate axonal growth. The molecular basis of this response has been extensively studied and a range of agents that promote or inhibit CAM stimulated axonal growth have now been identified. These studies have led to the suggestion that following homophilic and/or heterophilic interactions CAM specific signal transduction pathways are activated which are directly responsible for promotion of axonal growth. In this review we will suggest that the axonal growth response stimulated by three CAMs (NCAM, N-cadherin and L1) can be operationally divided into a number of phases. During the first phase homophilic and/or heterophilic binding between the CAMs expressed on the axonal growth cone and cellular substrate take place. This is followed by an interaction of the neuronal CAMs with the fibroblast growth factor receptor (FGFR), leading to receptor activation by autophosphorylation. This results in the recruitment and activation of additional effector molecules via interactions of their SH2 domains with the activated receptor. In this context the key event in terms of neurite outgrowth appears to be the activation of phospholipase C gamma (PLC gamma) which sets into motion a second messenger cascade that ultimately leads to a modification, most likely by phosphorylation, of cytoskeletal elements that are involved in growth cone motility.

Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor

Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor

A new route to thyrotoxicosis?

Human thyrocytes were found to synthesise and secrete the selenoenzyme extracellular glutathione peroxidase (E-GPX), a process which was controlled by the Ca2+/phosphoinositol second messenger cas cade. The potential involvement of thyroidal E-GPX in the regulation of thyroid hormone synthesis and in the protection of the thyrocyte from peroxidative damage is discussed.

Compartmentalized Production of Ceramide at the Cell Surface

Ceramide produced by the hydrolysis of sphingomyelin is an important cellular intermediate in hormone action. Here, we present evidence that interleukin 1 beta (IL-1 beta) binding to normal human fibroblasts initiates a lipid messenger cascade that takes place in a sphingomyelin-rich plasma membrane domain with the characteristics of caveolae. Hormone binding first stimulated the appearance of diacylglycerol (DAG) in a caveolearich membrane fraction isolated from whole cells. This was immediately followed by the loss of a resident population of sphingomyelin from the fraction and the concomitant appearance of ceramide. The ceramide produced in response to IL-1 beta blocked platelet-derived growth factor-stimulated DNA synthesis. IL-1 beta stimulated the appearance of DAG in other fractions from the same cell, but this DAG was not coupled to ceramide production. This indicates that ceramide production is highly compartmentalized at the cell surface. Since caveolae are known to be involved in membrane internalization, they may be essential for the delivery of ceramide to a site of action within the cell.

Calcium/calmodulin-sensitive adenylyl cyclase as an example of a molecular associative integrator

AbstractEvidence suggests that the Ca2+/calmodulin-sensitive adenylyl cyclase may play a key role in neural plasticity and learning in Aplysia, Drosophila, and mammals. This dually-regulated enzyme has been proposed as a possible site of stimulus convergence during associative learning. This commentary discusses the evidence that is required to demonstrate that a protein in a second messenger cascade actually functions as a molecular site of associative integration. It also addresses the issue of how a dually-regulated protein could contribute to the temporal pairing requirements of classical conditioning: that relationship between stimuli display both temporal contiguity and predictability.

Inflammatory mediators of pain

While sensory fibres normally respond to a range of physical and chemical stimuli their activity and metabolism are profoundly altered by a variety of mediators generated by tissue injury and inflammation. These include substances produced by damaged tissue, substances of vascular origin as well as substances released by afferent fibres themselves, sympathetic fibres and various immune cells. The effects of inflammatory mediators, to activate or sensitize afferent fibres, are produced by changing membrane ion channels which are coupled directly via receptors or more commonly are regulated through receptor-coupled second messenger cascades. These latter processes also have the potential to alter gene transcription and thereby induce long-term alterations in the biochemistry of sensory neurones. This can have far-reaching consequences as the expression of novel proteins for ion channels (Na channels) and receptors (capsaicin, NPY) as well as the induction of novel enzymes (i-NOS) can profoundly affect the properties of nociceptors and their ability to transmit pain signals. However, such changes may be targeted successfully for the development of new analgesic and anti-inflammatory agents.

ANF and angiotensin II interact via kinases in the proximal straight tubule.

Atrial natriuretic factor (ANF) inhibits fluid absorption (Jv) in the proximal straight tubule (PST) only after stimulation with angiotensin II (ANG II). To investigate ANF's dependency on ANG II for transport inhibition, we blocked and mimicked angiotensin's second messenger cascades and then examined ANF's ability to inhibit Jv. ANG II (10(-10) M)-stimulated Jv was 0.47 +/- 0.10 nl.mm-1. min-1. After ANF (10(-10) M) was added to the bath, Jv fell by approximately 40% (P < 0.05). ANG II stimulates Jv via activation of protein kinase C (PKC) and decreasing protein kinase A (PKA) activity. We inhibited PKA with H-89. In the presence of only H-89, Jv was 0.75 +/- 0.11 nl.mm-1.min-1. After ANF was added to the bath Jv fell by 30% (P < 0.05). Intracellular adenosine 3',5'-cyclic monophosphate content was not affected by ANF in the presence of ANG II. ANF could not inhibit Jv in the presence of ANG II and 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. KT-5823, a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase inhibitor, blocked the action of ANF on Jv (P > 0.30). PKC inhibition did not prevent the decrease in Jv induced by ANF. We conclude that ANF inhibits ANG II-induced stimulation of transport by a mechanism that requires phosphorylation mediated by cGMP-dependent protein kinase subsequent to a decrease of PKA activity.

Forskolin induces preproenkephalin and preprodynorphin mRNA in rat striatum as demonstrated by in situ hybridization histochemistry.

Cyclase response elements (CREs) are located in the promoter regions of several neuropeptide and immediate early genes. Activation of the adenylate cylase/cAMP second messenger cascade leads to phosphorylation of CRE-binding proteins (P-CREBs) which bind to CREs in the promoter regions of these genes and alter their rate of transcription. We have previously reported an increase in striatal immunoreactivity for P-CREB (phosphorylated on Ser-133) and Fos following intracerebroventricular (ICV) injection of H2O-soluble forskolin, a direct activator of adenylate cyclase. Because CREs are located in the promoter regions of the opioid peptide genes, preproenkephalin (PPE) and preprodynorphin (PPD), we investigated what effect continuous ICV infusion of H2O-soluble forskolin has on striatal PPE and PPD mRNA levels. Quantitative in situ hybridization histochemistry demonstrated that continuous activation of the adenylate cyclase/cAMP second messenger cascade results in a significant induction of striatal PPE and PPD mRNA at 6, 24, and 72 h. The sustained induction of striatal PPE and PPD mRNA indicates that pro-opioid gene transcription is not desensitized following 72 h of continuous adenylate cyclase activation. Continuous ICV infusion of 1, 9-dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not induce striatal PPE and PPD mRNA. These data are consistent with cAMP-dependent protein kinase-induced phosphorylation and binding of CREBs to CREs in the promoter regions of pro-opioid genes during sustained activation of adenylate cyclase.