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M J Smit

Publications and source records attributed to M J Smit.

At least 37 records · Page 2Linked to original sources
PubMed1998

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells.

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.

3T3 Cells↗
PubMed1998

Expression of Q227L-galphas in MCF-7 human breast cancer cells inhibits tumorigenesis.

The effects of expression of mutant (Q227L)-activated Galphas and elevation of cAMP on mitogen-activating protein kinase (MAPK) activity and the transformed phenotype were studied in the MCF-7 human mammary epithelial cell line. Elevation of cAMP partially inhibited the epidermal growth factor-stimulated DNA synthesis and the intrinsic MAPK (ERK-1 and ERK-2) of serum-starved MCF-7 cells. Addition of 8Br-cAMP or expression of mutant (Q227L)-activated Galphas in MCF-7 cells blocked the ability of these cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. 8Br-cAMP in the culture medium also blocked estrogen stimulation of MCF-7 cell proliferation in vitro. MCF-7 cells expressing Q227L-Galphas grew very slowly in vitro, and when these cells were injected s.c. into athymic mice implanted with estrogen pellets, the frequency of tumor formation was reduced greatly and the sizes of the tumors formed were much smaller than those in mice injected with MCF-7 cells that had been transfected with the empty vector. These results indicate that the intracellular levels of cAMP in transformed mammary epithelial cells can be a crucial factor in determining the expression of the transformed phenotype. Interactions between the Gs/adenylyl cyclase and MAPK-1,2 signaling pathways could be one mechanism by which expression of the transformed phenotype in mammary epithelial cells are regulated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Sensitization of the histamine H1 receptor by increased ligand affinity.

Histamine regulates a variety of physiological processes including inflammation, gastric acid secretion, and neurotransmission. The cellular response to histamine is subject to dynamic control, and exaggerated histamine reactivity in response to cysteinyl leukotrienes and other stimuli is important in a variety of different pathological conditions. The molecular mechanisms controlling histamine responsiveness are still unresolved. In investigating histamine responses in embryonic stem (ES5) and F9 embryonic carcinoma cells, we encountered a novel mechanism controlling the cellular reaction to histamine. Unstimulated cells displayed neither [3H]pyrilamine binding nor histamine-induced increases in cytosolic Ca2+ levels. Pretreatment of these cells, however, with leukotriene D4, leukotriene E4, serotonin, or fetal calf serum induced an immediate and transient ability of these cells to respond to histamine with an increase in cytosolic Ca2+ levels. This effect could be inhibited by pertussis toxin and was mimicked by GTP analogues. Importantly, the latter compounds also provoked immediate high affinity [3H]pyrilamine binding. We conclude that in these cells histamine responsiveness is directly controlled by pertussis toxin-sensitive G protein-coupled receptors, whose activation enables the H1 receptor to bind its ligand. These findings define a novel mechanism for regulating histamine H1 receptor activity and provide for the first time molecular insight into the mechanism by which cysteinyl leukotrienes and other external stimuli can increase histamine responsiveness.

Animals↗
PubMed1998

Agonist-independent regulation of constitutively active G-protein-coupled receptors.

G-protein-coupled receptors constitute one of the largest protein super-families in mammals. Since the cloning of the encoding genes, these important drug targets have been subjected to thorough biochemical and pharmacological studies. It has become clear that G-protein-coupled receptors not only transmit signals after stimulation by agonists but can also spontaneously couple to signal-transduction pathways. Recent findings show that constitutively active G-protein-coupled receptors can also be regulated in an agonist-independent manner, which has important implications for the interpretation of the actions of (inverse) agonists and the results of site-directed-mutagenesis studies.

Animals↗
PubMed1998

Constitutive activity and structural instability of the wild-type human H2 receptor.

Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine-induced increase in cAMP and the cimetidine-induced reduction of the basal cAMP level with apparent K(B) values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long-term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an up-regulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.

Animals↗
PubMed1998

Tibolone prevents atherosclerotic lesion formation in cholesterol-fed, ovariectomized rabbits.

Tibolone (Org OD14), a synthetic steroid with estrogenic and progestogenic/androgenic properties, is clinically effective for the treatment of climacteric symptoms and the prevention and treatment of osteoporosis in postmenopausal women. The effect on atherogenesis, however, is not known. In the current study, we investigated the effect of tibolone in comparison with that of estradiol and norethisterone acetate on atherogenesis in 140 ovariectomized New Zealand White rabbits that had been induced by an atherogenic diet (0.4% cholesterol, 20 weeks). Tibolone at 18, 6, or 2 mg/d orally completely prevented cholesterol accumulation and fatty streak formation in the aorta; the impairment of endothelium-dependent smooth muscle relaxation of the aorta; and complex lesion formation after endothelial denudation in the carotid artery. Tibolone also reduced the increased postovariectomy plasma lipid concentrations. Analysis of the results, however, indicated that a substantial part of the strong, beneficial effects were plasma lipid independent. Compared with subcutaneous estradiol decanoate (150 microgram once weekly) and oral 17beta-estradiol (4 mg/d), the effects of tibolone were more pronounced at equipotent uterotropic activity. Norethisterone acetate (1 mg/d) did not affect atherosclerotic lesion formation. There are no indications that the progestogenic/androgenic properties of tibolone counteracted its atheroprotective effect on the vessel wall. Therefore, tibolone has the intrinsic potential to be a compound that protects the arterial vessel wall against atherosclerotic processes.

Animals↗
PubMed1997

Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active G(S)-coupled receptors.

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin-induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK-293 and Sf9 cells or TSH receptor expression in COS-7 cells. The enhancement of forskolin-induced cAMP production seems to be a general characteristic of constitutively active G(S)-coupled receptors and can be very useful to study inverse agonism at wild-type receptors.

Adenylyl Cyclases↗
PubMed1997

Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Galphas stimulation.

Receptors activate adenylyl cyclases through the Galphas subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Galphas. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Galphas. Similar treatment of AC1 or AC2 did not affect Galphas stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Galphas stimulation. A peptide encoding the region 660-682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Galphas. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660-682 of AC6 is involved in regulation of signal transfer from Galphas. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.

Adenylyl Cyclases↗
PubMed1996

Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.

Animals↗
PubMed1996

Two distinct pathways for histamine H2 receptor down-regulation. H2 Leu124 --> Ala receptor mutant provides evidence for a cAMP-independent action of H2 agonists.

Pretreatment of Chinese hamster ovary cells expressing the histamine H2 receptor (CHOrH2 cells) with histamine resulted in a time-dependent (t1/2 approximately 7 h) and dose-dependent (EC50=18 nM) H2 receptor down-regulation measured as [125I]iodoaminopotentidine binding (44+/-10% down-regulation). Pretreatment of CHOrH2 cells with cholera toxin or forskolin also led to H2 receptor down-regulation. Forskolin time-dependently (t1/2 approximately 7 h) and dose-dependently (EC50 = 0.3 microM) induced H2 receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H2 receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O. Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H2 receptor from the Gs-protein, we introduced the Leu124 --> Ala mutation in the second intracellular loop of the H2 receptor. The H2 Leu124 --> Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH2 cells, H2 receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro.

Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i.p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage-colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i.p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth.

Animals↗
PubMed1996

The C terminal tail of the histamine H2 receptor contains positive and negative signals important for signal transduction and receptor down-regulation.

To examine the role of the C terminal tail in H2 receptor regulation, three cDNAs, encoding truncated histamine H2 receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2 receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2 antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gs protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H2 receptor down-regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2 receptor protein are involved in the cyclic AMP-dependent and independent pathways of H2 receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H2 receptor that act as positive and/or negative signals in H2 receptor signaling and down-regulation.

Amino Acid Sequence↗
PubMed1996

Regulation of the human histamine H1 receptor stably expressed in Chinese hamster ovary cells.

1. The human H1 receptor gene expressed in Chinese hamster ovary cells (CHOhumH1) encodes a classical histamine H1 receptor with a pharmacology similar to that of the H1 receptor found in guinea-pig cerebellum and the endogenously expressed human H1 receptor in 1321N1 astrocytoma cells as determined by [3H]-mepyramine binding studies. 2. In CHOhumH1 cells, histamine induced a concentration-dependent rise in inositol phosphates (EC50 2.23 +/- 0.97 microM) and a rapid increase of [Ca2+]i, followed by a sustained increase of [Ca2+]i upon addition of 100 microM histamine. 3. Short-term exposure of CHOhumH1 cells to histamine (100 microM) resulted in a decrease of subsequent histamine-induced Ca2+ responses. The histamine-induced desensitization appeared to be heterologous as the ATP-induced Ca2+ response was also found to be affected. 4. The process of heterologous histamine-induced desensitization of the Ca2+ response in CHOhumH1 cells can be ascribed to an alteration at the level of the intracellular Ca2+ pool, as the Ca2+ response of caffeine (10 mM), which releases Ca2+ from intracellular Ca2+ stores was also attenuated upon short-term histamine exposure. 5. In CHOhumH1 cells the PKC activator, PMA, was found to inhibit the histamine (100 microM)-induced Ca2+ response concentration-dependently (IC50 0.2 +/- 0.03 microM) as well as the ATP (100 microM)-induced Ca2+ response. However, this inhibition was only partial and less effective than histamine-pretreatment. Moreover, in CHOhumH1 cells PKC downregulation induced by long-term exposure to PMA (1 microM) did not affect the histamine-induced desensitization nor did pretreatment with the specific PKC inhibitor Ro-31-8220 (10 microM), indicating that in CHOhumH1 cells PKC is probably not involved in the heterologous desensitization. 6. Long-term treatment of CHOhumH1 cells with histamine or other H1 agonists resulted in a time- and concentration-dependent decrease in the number of H1 receptor binding sites (maximal reduction: 47 +/- 5%). 7. Long-term exposure of CHOhumH1 cells to ATP or PMA did not affect H1 receptor density. 8. Both histamine (100 microM)- and ATP (100 microM)-induced Ca2+ responses were affected upon long-term exposure of cells to histamine (100 microM), which might be explained by an alteration at a level distant from the receptor. 9. These results show that in CHOhumH1 cells the human histamine H1 receptor is susceptible to short-term and long-term receptor regulation in which PKC does not seem to play a role. The CHOhumH1 cells therefore provide an excellent model system for studying the mechanism(s) of PKC-independent H1 receptor regulation.

Animals↗
PubMed1995

Visualization of agonist-induced internalization of histamine H2 receptors.

Histamine H2 receptors were tagged at the N-terminus with the eight amino acid Flag epitope to allow the immunological identification of the receptor peptide with the monoclonal anti-Flag M2 antibody. The introduction of the epitope did not modify the binding of several H2 ligands to the H2 receptor, nor the ability of histamine to stimulate the H2 receptor mediated cAMP production in HEK-293 cells. Western blots revealed a major protein band of 57 +/- 1 kDa, whereas a second band of 31 +/- 1 kDa was probably the result of a proteolytic breakdown of the 57 kDa band. Immunofluorescence measurements of stably transfected HEK-293 cells revealed the presence of anti-Flag-immunoreactivity in the plasma membrane. This immunoreactivity completely disappeared after a one hour treatment with histamine. The receptor internalization was reversible and blocked by the endocytosis inhibitor phenylarsine oxide. Forskolin did not induce H2 receptor internalization, indicating that histamine causes H2 receptor internalization via a cAMP-independent pathway.

Amino Acid Sequence↗
PubMed1995

Lysine200 located in the fifth transmembrane domain of the histamine H1 receptor interacts with histamine but not with all H1 agonists.

Previously, we have shown that asparagine207 in the fifth transmembrane domain of the histamine H1 receptor is crucial for the binding of the N tau-nitrogen of the imidazole ring of histamine (Leurs et al., Biochem. Biophys. Res. Commun., 201, 295, 1994). In view of the potential interaction of the imidazole ring of histamine with a binding site, formed by asparagine207 and lysine200, we mutated lysine200 in the fifth transmembrane domain of the histamine H1 receptor to a non-functional alanine residue. This mutation did not affect the binding of the tested H1 receptor antagonists but resulted in a 5-fold lower affinity for histamine. The binding of other H1 receptor agonists was not affected. In stably transfected CHO cells histamine was 55-fold less effective in activating the H1Lys200Ala receptor (EC50 = 66 microM) compared to the wild type H1 receptor (EC50 = 1.2 microM). Receptor activation by the 2-methyl and the 2-(3-bromophenyl)-analogues however was hardly affected by the mutation, indicating that the 2-substituent probably prevents the interaction with the lysine200 residue. Finally, the Lys200Ala mutation reduced the production of [3H]inositol phosphates, stimulated by the non-imidazole H1 receptor agonist 2-pyridylethylamine. These data indicate that lysine200 interacts with the N pi-nitrogen of histamine and is important for the activation of the H1 receptor by histamine and the non-imidazole agonist 2-pyridylethylamine.

Amino Acid Sequence↗