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PubMed1996

N-palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids are selective competitive antagonists of the lysophosphatidic acid receptors.

Lysophosphatidic acid is the best characterized member of a lipid mediator family with growth factor-like activities that act through a class of G protein-coupled plasma membrane receptors. In Xenopus laevis oocytes, lysophosphatidate activates at least two pharmacologically distinct receptor subtypes distinguished by 1-acyl-sn-glycero-2,3-cyclic phosphate. Both of these naturally occurring ligands elicit oscillatory Cl- currents in the oocyte through G protein-coupled activation of the phosphoinositide/Ca2+ second messenger system, which in turn leads to the opening of Ca(2+)-activated Cl- channels. We developed an improved chemical synthesis and purification procedure for two N-acylated amino acid phosphates. N-Palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids inhibited the lysophosphatidate-activated Cl- currents with IC50 values of 5.4 +/- 0.7 and 6.5 +/- 1.5 nM at the high affinity site and 805 +/- 97 and 172 +/- 36 nM at the low affinity receptor site, respectively. In selective activation of the cyclic lysophosphatidate receptor, IC50 values of 330 +/- 30 and 490 +/- 40 nM were obtained, respectively. The D- and L-stereoisomers were equally effective when applied extracellularly. In contrast, they were ineffective when microinjected into the oocyte, indicating an extracellular site of inhibition. The inhibitors did not alter currents elicited by the different acetylcholine, serotonin, and glutamate receptors expressed heterologously in the oocyte. Pharmacological analysis of the results indicates that N-palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids are potent and specific competitive inhibitors of the lysophosphatidate receptors in the X. laevis oocyte.

Acetylcholine↗

PubMed1995

Platelet-derived growth factor causes sustained depletion of both inositol trisphosphate-sensitive and caffeine-sensitive intracellular calcium stores in vascular smooth muscle cells.

Since the platelet-derived growth factor (PDGF)-induced increase in cellular inositol 1,4,5-trisphosphate (InsP3) has been found to decay to basal levels soon after the onset of PDGF exposure, it has been argued that activation of Ca2+ release from intracellular stores must be similarly transient. The possibility remains, however, that PDGF-induced release of stored Ca2+ is initiated and sustained by other second-messenger systems. To test the hypothesis that PDGF-BB initiates sustained Ca2+ release from cellular stores, we performed 4-hour 45Ca effluxes on monolayers of A7r5 vascular smooth muscle cells in small, continuously perfused chambers. Isoform PDGF-BB (5 ng/mL for 30 minutes or 30 ng/mL for 15 minutes) was added to the perfusate beginning at 30 minutes of efflux. A dose-related increase in 45Ca release was sustained as long as PDGF-BB was present. Detailed kinetic analysis and nonlinear least-squares fitting of the experimental data revealed that (1) PDGF-BB induced sustained increases of 2.86-fold (5 ng/mL) and 6.50-fold (30 ng/mL) in the rate constant governing Ca2+ release from intracellular stores, (2) the apparent Km for this effect was 13.4 +/- 1.31 ng PDGF-BB/mL, and (3) the entire agonist-releasable Ca2+ store (presumably sarcoplasmic reticulum) is sensitive to PDGF-BB. These data indicate that PDGF-BB causes a sustained depletion of intracellular Ca2+ stores by means of sustained activation of Ca2+ release and suggest that intraorganellar Ca2+ may be one of the signals that mediates long-term smooth muscle responses to PDGF.

Animals↗

PubMed1992

Protein kinases and the androgen-induced proliferation of accessory sex organ smooth muscle.

The possible role of second messenger systems in androgen-dependent smooth muscle proliferation was investigated. Focusing on the hormone-sensitive guinea pig seminal vesicle, we analyzed changes in protein kinase C (PKC) and cAMP-dependent type I and II protein kinases during the androgen-dependent smooth muscle proliferation of puberty, as well as in the transition to the nonproliferative state of the adult. The androgenic sensitivity of the cAMP-dependent type I and II protein kinases and the cAMP-dependent phosphorylations of soluble muscle proteins did not correlate with the qualitative change in the androgenic sensitivity of the prepubertal vs. adult animals. In contrast to the cAMP-dependent protein kinases, regulation of the soluble and particulate forms of PKC corresponded to the androgen-induced smooth muscle proliferation. That is, in the seminal vesicle muscle of prepubertal castrated animals, androgen treatment reduced both the soluble and particulate forms of PKC during the increase in smooth muscle DNA synthesis, and in adult seminal vesicle smooth muscle, which was resistant to androgen-induced proliferation, both forms of the enzyme were resistant to androgenic stimulation. It is concluded that PKC may be a component of an autocrine mitogenic mechanism involved in the coupling and uncoupling of androgen-induced smooth muscle proliferation.

Androgens↗

PubMed1995

Absence of D2S dopamine receptor in the prolactin-secreting MMQ pituitary clone: characterization of a wild D2L receptor coupled to native transduction mechanisms.

We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactin- and ACTH-secreting Buffalo rat 7315 alpha pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the well-known process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding resulted in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0.055 +/- 0.002 nM and a Bmax of 27 +/- 3.5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (-)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.

Adenylyl Cyclases↗

PubMed1990

Inhibition of muscarinic-coupled phosphoinositide hydrolysis by N-methyl-D-aspartate is dependent on depolarization via channel activation.

The intent of this work was to elucidate the mechanism by which N-methyl-D-aspartate (NMDA) receptor agonists inhibit a second messenger system, namely, the stimulation of phosphoinositide (PI) hydrolysis activated by muscarinic cholinergic receptor agonists. NMDA inhibited cholinergic stimulation of PI hydrolysis in a dose- and time-dependent manner. NMDA exerts this effect indirectly through channel activation, because both MK-801 and N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) prevented this action. Prevention of the NMDA effect by removal of sodium, but not calcium, from the incubation buffer suggested that depolarization may be the responsible mechanism. Depolarization alone proved sufficient to inhibit cholinergic activation of PI hydrolysis, because both veratridine and an elevated extracellular potassium level inhibited cholinergic stimulation of PI hydrolysis. The effect of NMDA appeared to require sodium flux through NMDA channels rather than through voltage-dependent sodium channels, because tetrodotoxin failed to inhibit the effect of NMDA. In correlative electrophysiologic experiments, NMDA profoundly inhibited evoked excitatory postsynaptic potentials and population action potentials of CA1 neurons, an effect almost certainly due to depolarization. The dose and time course of the electrophysiologic effects correlated well with the biochemical effects. Taken together, the data support the assertion that NMDA receptor activation inhibits PI hydrolysis by depolarization mediated by sodium flux through NMDA channels.

Animals↗

PubMed1996

Modulation of membrane currents by cyclic AMP in cleavage-arrested Drosophila neurons.

A number of Drosophila learning mutants have defective intracellular second-messenger systems. In an effort to develop techniques that will allow direct measurement of the effects of these mutations on whole-cell neuronal membrane currents, the perforated-patch whole-cell (PPWC) technique has been applied to cleavage-arrested cultured embryonic Drosophila neurons. This technique permits the measurement of membrane currents without disturbing the intracellular environment. As a result of the maintenance of the intracellular environment, Drosophila neuron currents are found to be much more stable than when measured using the conventional whole-cell (CWC) patch-clamp technique. Ca2+ channel currents, which typically 'wash out' within a few minutes of the beginning of CWC recording, are stable for the duration of the seal (tens of minutes) when measured using the PPWC technique. Since the learning mutations dunce and rutabaga disrupt cyclic AMP signalling, the action of externally applied dibutyryl cyclic AMP (db-cAMP) and theophylline on Ca2+ and K+ channel currents were studied. db-cAMP and theophylline enhanced the Ba2+ current, carried by Ca2+ channels, but had no effect on the K+ current in the cleavage-arrested neurons. However, the large variability and reduction in density of Ba2+ and K+ currents raise questions about the suitability of using these cleavage-arrested cells as models for Drosophila neurons.

Animals↗

PubMed1994

Intracellular calcium signaling induced by thapsigargin in excitable and inexcitable cells.

Signaling between intracellular Ca2+ stores and cell membrane channels or transporters is important to Ca(2+)-based second messenger systems. Two hypotheses, the capacitative and the Ca(2+)-induced Ca(2+)-influx models have been proposed to explain aspects of this signaling. In this study, we examined the applicability of these models in neuroendocrine (PC12), neuronal (dorsal root ganglion), immune (spleen), and fibroblast (3T3) cells. We used thapsigargin (TPG) to deplete specific intracellular Ca2+ stores and to increase the cytoplasmic Ca2+ concentration ([Ca2+]), and Ca2+ free medium to prevent Ca2+ influx and lower cytoplasmic [Ca2+]. We demonstrate that, although TPG causes an increase of [Ca2+]i in all cells examined, the subsequent stimulation of Ca2+ influx varies from high in spleen, to moderate in 3T3 and PC12, to undetectable in DRG cells. All cell types exhibited Ca2+ influx when Ca2+ was added to the medium following an exposure to Ca(2+)-free medium. Without added provisions, the two aforementioned hypotheses are inadequate in explaining the TPG-induced Ca(2+)-influx in all cell types. These results support the hypothesis of the existence of unique Ca2+ channels or transporters in spleen cells that operate subsequent to TPG treatment and are distinct from the voltage-gated Ca2+ channels and Ca(2+)-activated non-selective cation channels present in excitable cells.

3T3 Cells↗

PubMed1995

Stimulation of angiotensin II AT1 receptors in rat median eminence increases phosphoinositide hydrolysis.

The aim of our study was to determine the second messenger systems for angiotensin II in the rat median eminence. Angiotensin II AT1 receptors are highly expressed in the median eminence and binding is selectively inhibited by the guanine nucleotide GTP gamma S, indicating possible coupling to G-proteins. In male rats, angiotensin II increased phosphatidylinositol hydrolysis about 45% over basal values, with an EC50 of about 2.7 nM. This effect was antagonized by 10 microM losartan, the selective AT1 antagonist, but not by the AT2 competitor PD 123319. Conversely, angiotensin II, 1 microM, did not alter basal or forskolin-stimulated cAMP production, and failed to influence cGMP production. These results support a role for angiotensin II, through stimulation of AT1 receptors and increased phosphatidylinositol hydrolysis, in the median eminence. Angiotensin II increased the phosphatidylinositol hydrolysis not only in male rats but also in ovariectomized rats, with or without estrogen-progesterone replacement. However, angiotensin II (up to 1 microM) failed to increase the phosphatidylinositol hydrolysis in randomly selected intact female rats. Estrogen treatment did not alter the number or affinity of median eminence AT1 receptors in ovariectomized rats. The increase in phosphatidylinositol hydrolysis resulting from stimulation of median eminence AT1 receptors appears to be sexually dimorphic, but hormonal manipulations failed to point to a role for reproductive hormones in this phenomenon.

Angiotensin II↗

PubMed1996

Noradrenergic potentiation of cerebellar Purkinje cell responses to GABA: cyclic AMP as intracellular intermediary.

Norepinephrine and the beta-adrenergic receptor agonist, isoproterenol, have been shown to potentiate the amplitude of GABAA receptor-mediated whole-cell current responses in Purkinje cells acutely dissociated from the rat cerebellum. However, the steps leading from the activation of beta-adrenergic receptors to the modulation of GABAA receptor remain to be delineated. This study tested the hypothesis that a sequelae of intracellular intermediaries involving the cyclic AMP second messenger system serves as the subcellular link to promote this heteroreceptor interaction. Exposure to cholera toxin, but not to pertussis toxin, increased the amplitude of GABA-activated current responses in acutely dissociated Purkinje cells. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) also resulted in a time- and dose-dependent augmentation of the response to GABA. while guanosine 5'-O-(2-thiodiphosphate) blocked the norepinephrine-mediated facilitation. A positive modulation of the current response to GABA was observed following intracellular delivery of cyclic AMP or the catalytic subunit of the cyclic AMP-dependent protein kinase. Furthermore, the norepinephrine-induced potentiation of the GABA-activated current response was prevented in the presence of the Rp isomer of cyclic AMP, the regulatory subunit of cyclic AMP-dependent protein kinase and an inhibitor of cyclic AMP-dependent protein kinase. These findings led to the formulation of a working model in which activation of the beta-adrenergic receptor triggers a Gs-protein-mediated transduction cascade in cerebellar Purkinje cells which activates adenylate cyclase, resulting in a rise in intracellular levels of cyclic AMP, increased phosphorylating activity by cyclic AMP-dependent protein kinase and, ultimately, a potentiation of GABAA receptor function.

Adenylate Cyclase Toxin↗

PubMed1991

Phosphoinositides and synaptic function in NG108-15 neuroblastoma x glioma hybrid cells.

1. The second-messengers system of bradykinin (BK) receptors was examined in NG108-15 neuroblastoma x glioma hybrid cells. 2. An application of BK induced an immediate outward (K+) current and acetylcholine (ACh) release, which are generated through inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ ions. 3. Application of phorbol dibutyrate (a protein kinase C activator) produced a voltage-dependent inward current and inhibited another K+ (M)-current. 4. A similar current response has been produced by ACh in NG108-15 cells transfected with rodent muscarinic ACh receptor I and III subtype genes. 5. These results suggest a dual and time-dependent role for these two intracellular messengers in the control of neuronal signalling by BK and ACh.

Acetylcholine↗

PubMed1994

Characterization of a functional angiotensin II receptor in Xenopus laevis heart.

High-affinity (104 +/- 18 pmol/l) and high-density (204 +/- 25 fmol/mg) angiotensin II (AII) binding sites have been identified in Xenopus laevis heart membranes. Competition binding of [125I]Sar1,Ile8 angiotensin (SIA) to these receptors by peptide analogs selective for the mammalian AII receptor subtypes AT1 and AT2 suggested that the amphibian AII binding sites were more closely related to the AT1 receptor subtype. Also in common with AT1 receptors, dithiothreitol and GTP gamma S inhibited [125I]SIA binding to Xenopus heart receptors, exhibiting IC50 values of 600 and 0.95 mumol/l, respectively. In addition, Xenopus oocytes injected with Xenopus heart mRNA were capable of mobilizing calcium when exposed to AII, demonstrating that Xenopus AII receptors are functionally linked to a second-messenger system similar to that coupled to mammalian AT1 receptors. However, in contrast to both AT1 and AT2 receptor subtypes, nonpeptide antagonists DUP 753 and SK&F 108566 (AT1 receptor selective) and PD123319 (AT2 selective) did not bind the Xenopus AII receptors, thus establishing that the amphibian receptors were pharmacologically unique. Together, these results demonstrate that Xenopus heart AII receptors are functionally similar to mammalian AT1 receptors but are pharmacologically distinct from both AT1 and AT2 receptors.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1990

Nerve growth factor and fibroblast growth factor regulate neurite outgrowth and gene expression in PC12 cells via both protein kinase C- and cAMP-independent mechanisms.

Nerve growth factor (NGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) promote the survival and differentiation of a variety of peripheral and central neurons. The signal transduction mechanisms that mediate the actions of these factors in neuronal cells are not well understood. We examined the effect of a deficiency in protein kinase C (PKC) and/or cAMP second messenger systems on the actions of NGF, aFGF, and bFGF in the pheochromocytoma (PC12) cell line. Activation of PKC was not required for NGF, aFGF, and bFGF to maximally induce ornithine decarboxylase (ODC), transcription of the early response genes, d2 and d5, or neurite outgrowth. In a PC12 cell mutant that is deficient in cAMP responsiveness (A126-1B2), all three growth factors maximally induced the transcription of d5 and neurite outgrowth, but aFGF and bFGF did not induce significant increases in ODC. NGF and aFGF maximally induced the transcription of d2 in A126-1B2 cells, but bFGF-induced d2 transcription was attenuated. NGF, aFGF, and bFGF maximally induced neurite outgrowth and d5 transcription in A126 cells that were made deficient in PKC. The d2 transcriptional response was substantially reduced in cells deficient in both PKC and cAMP responsiveness. These observations lead us to conclude that (a) cAMP- and PKC-dependent events are, at least in part, causally linked to NGF, aFGF, and bFGF induction of both ODC and transcription of d2 and may control functionally redundant pathways; (b) NGF, aFGF, and bFGF can elicit neurite outgrowth and increase transcription of d2 and d5 in PC12 cells via mechanisms that are independent of both PKC and cAMP; (c) NGF, aFGF, and bFGF can induce ODC in the absence of PKC; and (d) aFGF and bFGF require cAMP responsiveness to induce ODC in PC12 cells.

Adrenal Gland Neoplasms↗

PubMed1993

Postischemic changes of intracellular second messengers in the gerbil brain after long-term survival: an autoradiographic study.

Receptor autoradiographic and histological techniques were used to investigate the long-term changes that occur in the gerbil brain following the induction of transient cerebral ischemia. Transient ischemia was induced for 3 and 10 min, and animals were allowed to survive for eight months. Autoradiographic analysis of second messenger systems showed that 3-min ischemia caused a significant reduction in [3H]inositol-1,4,5-trisphosphate binding in the hippocampal CA1 sector, whereas the alteration in [3H]phorbol 12,13-dibutyrate, [3H]forskolin and [3H] cyclic-AMP bindings was not found in this region. In the striatum, 3-min ischemia caused no significant alteration in [3H]phorbol 12,13-dibutyrate, [3H]inositol-1,4,5-trisphosphate and [3H]forskolin binding sites, whereas the [3H]cyclic-AMP binding showed a significant elevation. The thalamus exhibited a significant elevation only in the [3H]inositol-1,4,5-trisphosphate binding sites. Following 10-min ischemia, [3H]phorbol 12,13-dibutyrate, [3H]inositol-1,4,5-trisphosphate and [3H]cyclic-AMP binding sites revealed a significant reduction in the hippocampus, whereas the [3H]forskolin binding showed a significant elevation in this area. In the striatum, 10-min ischemia caused no significant alteration in [3H]phorbol 12,13-dibutyrate, [3H]inositol-1,4,5-trisphosphate and [3H]cyclic-AMP binding sites. However, marked reduction in the [3H]forskolin binding was seen in the striatum. Furthermore, the substantia nigra also exhibited a significant reduction in [3H]forskolin binding. Histological studies suggested that 3-min ischemia can produce severe neuronal damage and mild shrinkage to the hippocampal CA1 sector. They also showed that 10-min ischemia can cause severe tissue shrinkage and severe neuronal damage in the hippocampal CA1 sector and hippocampal CA3 sector. Thus, the hippocampal damage following ischemia was not static but progressive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Specific identification and subcellular localization of three calmodulin-binding proteins in the rat gonadotrope: spectrin, caldesmon, and calcineurin.

In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (Mr greater than 205,000), caldesmon (Mr 84,000), and the alpha subunit of calcineurin (Mr 60,000). The Mr greater than 205,000 and Mr 60,000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an Mr 84,000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (Mr 150,000). Like caldesmon from other sources, the Mr 84,000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.

Animals↗

PubMed1993

Cyclic adenosine monophosphate response in primary and subcultured bladder epithelial cells: inhibition by 12-O-tetradecanoylphorbol-13-acetate.

Primary and first-passage dog urothelial cells (DUC and DUC-P1, respectively) exhibited an active catalytic subunit for the cyclic adenosine monophosphate (cAMP) second messenger system. Dramatic increases in cAMP levels were observed following the addition of forskolin, which elicited a time- and dose-response--dependent increase in cAMP levels. Increases in intracellular cAMP levels preceded media increases in cyclic nucleotide levels and were observed at the earliest time examined (5 minutes). The lowest effective concentration of forskolin was between 1 and 10 mumol/L. cAMP level increases as large as 20- to 100-fold were observed in cells and media. Preincubation of primary and subcultured cells with 0.1 mumol/L 12-O-tetradecanoylphorbol-13-acetate (TPA) for 60 minutes reduced the magnitude of the forskolin-induced increase in cAMP levels. To determine the mechanism by which TPA elicits its effect in primary cultures, the following test agents were used: 1.0 mumol/L staurosporine and 25 mumol/L sphingosine, protein kinase C inhibitors; 35 mumol/L cycloheximide, a protein synthesis inhibitor; 3.0 mumol/L indomethacin, an inhibitor of prostaglandin synthesis; and 0.5 mmol/L RO-20-1724, a cyclic nucleotide phosphodiesterase inhibitor. Staurosporine and sphingosine were the only agents that prevented the effect of TPA. The specificity of the TPA effect was evaluated with the following test agents: 0.2 nmol/L epidermal growth factor (EGF), 0.1 mumol/L 4 alpha-TPA (a stereoisomer of TPA), or 1.0 mumol/L A23187. In contrast to TPA, none of these agents reduced forskolin-mediated increases in cAMP. Results indicate forskolin cAMP responsiveness and regulation of this response by TPA in both primary and subcultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

PubMed1992

Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF.

The effects of low density lipoprotein (LDL) and high density lipoprotein (HDL3) on second messenger systems were investigated in cultured human vascular smooth muscle cells (VSMC) and compared with those of angiotensin II (Ang II) and platelet-derived growth factor (PDGF-BB). Phosphoinositide metabolism was studied in myo-[2-3H]-inositol prelabelled VSMC using high performance liquid anion-exchange chromatography. The spectra of inositol phosphate isomers increased after stimulation with either Ang II, LDL, HDL3 or PDGF-BB were qualitatively identical. Major increases occurred in 4-IP1, 1,4-IP2, 1,3,4-IP3 and 1,3,4,5-IP4. These are metabolic conversion products of 1,4,5-IP3 for which only a minor increase was found. Thus lipoproteins, like Ang II and PDGF-BB, activate polyphosphatidylinositol-specific phospholipase C. Intracellular Ca2+ concentrations ([Ca2+]i) were studied in fura-2 loaded VSMC. In monolayer cultures LDL and HDL3 increased [Ca2+]i with kinetics comparable to those for Ang II. Relative to the effects of these agonists, the PDGF-BB-induced increase in [Ca2+]i was slower in onset and the decay from peak [Ca2+]i levels more gradual. Fluorescence recordings from single cells exposed to LDL and HDL3 revealed a prolonged series of transient oscillations of [Ca2+]i, a phenomenon typical for calcium-mobilizing hormones. Additionally, as found for Ang II, preincubation of VSMC with either phorbol 12-myristate, 13-acetate, forskolin or 8-bromo-cyclic GMP inhibited LDL- and HDL-induced accumulation of [3H]-inositol monophosphate. We propose that LDL and HDL3 stimulate signal transduction in VSMC via mechanisms analogous to those of Ca(2+)-mobilizing hormones.

Angiotensin II↗

PubMed1995

Follicle stimulating hormone (FSH) stimulates transferrin gene transcription in rat Sertoli cells: cis and trans-acting elements involved in FSH action via cyclic adenosine 3',5'-monophosphate on the transferrin gene.

FSH is a major regulator of transferrin (Tf) production in the testis. FSH effects on Sertoli cell Tf production are believed to be mediated, at least in part, via cAMP second messenger system. Previously, it has been shown that FSH and (Bu)2cAMP stimulate Tf mRNA levels. This study examines the effect of cAMP and FSH on Tf gene transcription using run-on assays. These data demonstrate rapid induction of Tf gene by (Bu)2cAMP (2.3-fold) and FSH (2.8-fold) within 30 min and 2 h, respectively. Furthermore, the ability of (Bu)2cAMP and FSH to drive the transcription of chimeric constructs containing a 0.6-kilobase segment of the 5'-regulatory region of the human Tf gene coupled to a chloramphenicol acetyltransferase (CAT) was examined. Deletion analysis indicated that the sequence -100/-52 base pairs is required for the cAMP-dependent transcription. This sequence shows no homology to that of the consensus cAMP-regulatory element (CRE). However, cotransfection experiments with a CRE-binding protein (CREB) expression vector revealed a basal induction of the Tf transcriptional activity as well as a synergistic activation of CREB and (Bu)2cAMP. Expression of KCREB, a dominant negative mutant form of CREB, completely blocked the cAMP induction of the -100+39Tf-CAT construct. This region contains two functional regions PRI and PRII. Gel shift assay with nuclear proteins from Sertoli cells using the PRII and PRI probes showed that the band shifts formed by PRII were competitive complexes with CRE, and a CREB antiserum retarded the migration of nuclear Sertoli cells proteins. We conclude that CREB is implicated in the FSH regulation on the Tf gene in Sertoli cells.

Animals↗

PubMed1989

Induction of protooncogene fos by extracellular signals in primary glial cell cultures.

In the present study various extracellular factors, acting through different second messenger systems, were examined for their capacity to increase the level of c-fos mRNA in primary glial cell cultures. In particular EGF, 12-O-tetradecanoylphorbol 13-acetate, the beta-adrenergic agonist isoproterenol, and the glutamate agonists, ibotenic and quisqualic acid, were studied. All the extracellular stimuli tested induced a rapid and transient increase in c-fos mRNA level in glial cell cultures regardless of the signal transduction pathway and the final effect on cell proliferation.

Animals↗