Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells

Effects of endothelin on DNA synthesis were investigated in two clones of vascular smooth muscle cells, 1YB4 and A7r5. The peptide stimulated DNA synthesis in both clones with apparent EC50 of less than 1 ng/ml. More than 17 h was required before initiating endothelin-stimulated DNA synthesis. The platelet-derived growth factor at a concentration which had no effects by itself on DNA synthesis enhanced the effect of low concentrations of endothelin. A calcium antagonist, nifedipine, inhibited endothelin-induced DNA synthesis. These data suggest that endothelin stimulates DNA synthesis in vascular smooth muscle cells through nifedipine-sensitive mechanisms that can be modulated by platelet-derived growth factor.     

Activation of S6 kinase in cultured vascular smooth muscle cells by submitogenic levels of thrombospondin

Purified human platelet thrombospondin was shown to activate S6 kinase in cultured vascular smooth muscle cells in a dose- (1-9 micrograms/ml) and time-dependent manner. Down regulation of epidermal growth factor and somatomedin C receptors by prior treatment of cells with their respective growth factors did not reduce this effect. Kinase activation by thrombospondin was only marginally reduced in the presence of platelet-derived growth factor specific antibody at levels that totally inhibited platelet-derived growth factor (5 ng/ml) induced activation. Additionally, thrombospondin elicits a rapid dose-dependent phosphoinositide turnover response analogous to that of platelet-derived growth factor, epidermal growth factor and somatomedin C. Prior treatment of cells with phorbol ester for 48 hrs in serum-free culture medium resulted in a small enhancement of S6 kinase activation by thrombospondin and the above mentioned growth factors but a complete loss in the ability of phorbol ester to activate this enzyme. These findings with cultured smooth muscle cells suggest a growth factor-like role for thrombospondin.     

Coupling between inositol phosphate formation and DNA synthesis in smooth muscle cells stimulated with neurokinin A

The two mammalian neuropeptides substance P (SP) and neurokinin A (NKA) have been demonstrated to stimulate DNA synthesis in connective tissue cells, suggesting that peripheral neurons may play a role in development and tissue regeneration. In this study we have tried to identify intracellular messengers required for SP- and NKA-induced DNA synthesis. SP and NKA, as well as platelet-derived growth factor (PDGF) stimulated formation of inositol phosphates in smooth muscle cells (SMC), whereas no effect on inositol phosphates formation occurred in response to nonmitogenic neuropeptides. Pretreatment of the cells with pertussis toxin markedly decreased DNA synthesis induced by NKA. This toxin inhibits formation of inositol phosphates by acting on a regulatory G-protein. Calcium and calmodulin antagonists also inhibited NKA-induced DNA synthesis. These results imply that the mitogenic signal(s) produced by activated neuropeptide receptors involves formation of inositol phosphate and activation of a calcium/calmodulin dependent process. We further report that other neuropeptides occurring in peripheral neurons, i.e., vasoactive intestinal polypeptide, calcitonin gene-related peptide, neuropeptide Y, somatostatin, or cholecystokinin, are without growth-stimulatory effect on cultured SMC.     

Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells : Differences between cells from young and adult animals

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and proliferation in cultures of arterial smooth muscle cells obtained from young and adult rats, respectively, were measured. Addition of 10-20 ng/ml of PDGF to medium MCDB 104 induced DNA synthesis in quiescent cultures of cells from young animals to a similar extent as 10-20% whole blood serum (WBS). PDGF further stimulated proliferation of the cells in medium MCDB 104, although less markedly than 10% WBS. Antibodies against PDGF partially inhibited the growth response after stimulation with serum. This shows that PDGF is a major growth factor in serum for these cells and that PDGF can promote entrance into and passage through S phase and mitosis independent o plasma factors. Cells from adult animals were also found to respond to PDGF, although a higher concentration (25 ng/ml) was required to obtain a maximum effect. These cells, however, responded better than cells from young animals to stimulation with serum. Further, antibodies against PDGF did not inhibit the growth-stimulatory effect of serum to any appreciable extent. Thus, serum contains growth factors other than PDGF that stimulate preferentiaLly the proliferation of smooth muscle cells from adult animals.     

Adenosine receptors, cyclic AMP accumulation, and DNA-synthesis in aortic smooth muscle cell cultures of adult and neonatal rats.

The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.     

Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.     

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.     

Selective inhibition of β2-adrenergic receptor-mediated cAMP generation by activation of the P2Y2 receptor in mouse pineal gland tumor cells

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.     

Pertussis Toxin Enhances Proenkephalin Synthesis in Bovine Chromaffin Cells

Abstract: The synthesis of the neuropeptide precursor proenkephalin was measured in bovine adrenal Chromaffin cells following radiolabeling with [³⁵S]methionine. Treatment of Chromaffin cells with pertussis toxin (100 ng/ml) approximately doubled proenkephalin synthesis without altering total protein synthesis. Pertussis toxin pretreat-ment also increased proenkephalin synthesis in chromaf-fin cells exposed to vasoactive intestinal peptide (VIP) and 3-isobutyl-1 -methylxanthine (IBMX). Combinations of IBMX plus nicotine, VIP, or histamine also synergistically enhanced proenkephalin synthesis, with no further elevation when the cells were also pretreated with pertussis toxin. The action of forskolin, a direct activator of adenyl-ate cyclase, on proenkephalin synthesis was similarly potentiated by pertussis toxin or IBMX, presumably reflecting the abilities of both the toxin and this phosphodiester-ase inhibitor to enhance the cyclic AMP response to forskolin. In contrast, increased synthesis of proenkephalin in response to phorbol esters was not affected by pertussis toxin treatment. These results suggest that pertussis toxin potentiates proenkephalin synthesis primarily through inactivation of guanine nucleotide-binding proteins that inhibit adenylate cyclase, although other signaling pathways may also be involved.     

The epidermal growth factor receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable guanine nucleotide-binding regulatory protein in cultured rat inner medullary collecting tubule cells

Studies were performed to examine a potential role for a guanine nucleotide-binding protein in epidermal growth factor (EGF)-stimulated phospholipase A2 (PLA2) activity. EGF increased prostaglandin E2 (PGE2) production in intact or saponin-permeabilized rat inner medullary collecting tubule (RIMCT) cells. Incubation of permeabilized cells with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) enhanced and with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) inhibited the response to EGF. GDP beta S had no effect on ionomycin-stimulated PGE2 production. Exposure of intact cells to 25 mM NaF + 10 microM AlCl3 enhanced both basal and EGF-stimulated PGE2 production. Pertussis toxin ADP-ribosylated a 41-kDa protein in RIMCT cell membranes. Pretreatment of cells with pertussis toxin (100 ng/ml for 16 h) eliminated the response to EGF in intact cells and the response to EGF + GTP gamma S in permeabilized cells. Pertussis toxin had no effect on the response to ionomycin. The effect of pertussis toxin was not due to alterations in cAMP as cellular cAMP levels were unaffected by pertussis toxin both in the basal state and in the presence of EGF. PGE2 production in response to EGF was not transduced by a G protein coupled to phospholipase C (PLC) as neomycin, which inhibited PLC, did not decrease EGF-stimulated PGE2 production. Also, PGE2 production was not increased by inositol trisphosphate and did not require the presence of extracellular Ca2+. In contrast to EGF-stimulated PLC activity, stimulation of PLA2 by EGF was not susceptible to inhibition by phorbol 12-myristate 13-acetate. These results clearly demonstrate the existence of a PLA2-specific pertussis toxin-inhibitable guanine nucleotide-binding protein coupled to the EGF receptor in RIMCT cells.     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.     

Thrombin-induced proliferation and expression of platelet-derived growth factor-A chain gene in human vascular smooth muscle cells.

Treatment of human vascular smooth muscle cells (SMC) with human alpha-thrombin greatly increased DNA synthesis and cell proliferation. Both the integrity of the catalytic site and that of the anion binding exosite were required for expression of this activity. Experiments employing Northerns indicated induction of c-fos expression as well as a time-dependent induction of platelet-derived growth factor-A (PDGF-A) gene by thrombin. The thrombin mitogenic activity was potentiated by PDGF-BB, insulin and the vasoconstrictor peptide endothelin-1 suggesting synergism by convergence of intracellular growth-promoting signals. SMC treatment with pertussis toxin and forskolin indicated that the mitogenic activity of thrombin may be induced via signal transduction mechanism(s) involving changes in cAMP levels and activation of a Gi-like protein. These results suggest that thrombin may play a functional role in the regulation of human vascular SMC proliferation.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.     

Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L-cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP-elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP-elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP-stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid-insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose-response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP-stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP-elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.     

Pertussis toxin inhibits EGF-, phorbol ester- and insulin-stimulated DNA synthesis in BALB/c3T3 cells: evidence for post-receptor activation of Gi alpha

The contribution of the GTP-binding protein, Gi, to EGF, phorbol dibutyrate (PdBu)-, and insulin-stimulated DNA synthesis was examined in BALB/c3T3 cells. Pertussis toxin inhibited DNA synthesis by each agonist, particularly at suboptimal agonist concentrations, but the inhibition could be partially overcome with higher agonist concentrations and combinations of these agonists. This suggested that (1) some, but not all, of the mitogenic signals for all three agonists were transduced by Gi (2) Gi may be activated by post-receptor mechanisms involving protein kinase C. Gi alpha-specific antibodies and ADP-ribosylation by pertussis toxin using 32P-NAD each labelled a single protein band, representing one or more species of Gi alpha. Pertussis toxin treatment increased the synthesis of Gi alpha. These results are discussed in relation to possible direct effects of Gi alpha on nuclear control during division.     

Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms

Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.