Short Periods of Hypoxia Upregulate Sphingosine Kinase 1 and Increase Vasodilation of Arteries to Sphingosine 1-Phosphate (S1P) via S1P3.

Sphingosine kinase [(SK), isoforms SK1 and SK2] catalyzes the formation of the bioactive lipid, sphingosine 1-phosphate (S1P). This can be exported from cells and bind to S1P receptors to modulate vascular function. We investigated the effect of short-term hypoxia on SK1 expression and the response of arteries to S1P. SK1 expression in rat aortic and coronary artery endothelial cells was studied using immunofluorescence and confocal microscopy. Responses of rat aortic rings were studied using wire myography and reversible hypoxia induced by bubbling myography chambers with 95% N2:5% CO2 Inhibitors were added 30 minutes before induction of hypoxia. S1P induced endothelium-dependent vasodilation via activation of S1P3 receptors and generation of nitric oxide. Hypoxia significantly increased relaxation to S1P and this was attenuated by (2R)-1-[[(4-[[3-methyl-5-[(phenylsulfonyl)methyl] phenoxy]methyl]phenyl]methyl]-2-pyrrolidinemethanol [(PF-543), SK1 inhibitor] but not (R)-FTY720 methyl ether [(ROMe), SK2 inhibitor]. Hypoxia also increased vessel contractility to the thromboxane mimetic, 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α, which was further increased by PF-543 and ROMe. Hypoxia upregulated SK1 expression in aortic and coronary artery endothelial cells and this was blocked by PF-543 and 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole [(SKi), SK1/2 inhibitor]. The effects of PF-543 and SKi were associated with increased proteasomal/lysosomal degradation of SK1. A short period of hypoxia increases the expression of SK1, which may generate S1P to oppose vessel contraction. Under hypoxic conditions, upregulation of SK1 is likely to lead to increased export of S1P from the cell and vasodilation via activation of endothelial S1P3 receptors. These data have significance for perfusion of tissue during episodes of ischemia.

Expression of sphingosine 1-phosphate receptor 4 and sphingosine kinase 1 is associated with outcome in oestrogen receptor-negative breast cancer.

BACKGROUND: We previously reported that sphingosine 1-phosphate receptor 4 (S1P(4)) is expressed and stimulates the ERK-1/2 pathway via a human epidermal growth factor receptor 2 (HER2)-dependent mechanism in oestrogen receptor-negative (ER(-)) MDA-MB-453 breast cancer cells. METHODS: Clinical relevance of S1P(4) and sphingosine kinase 1 (SK1, which catalyses the formation of S1P) was assessed in a cohort of 140 ER(-) breast tumours by immunohistochemistry (IHC) and the weighted histoscore method. Additional evidence for a functional interaction between S1P(4) and SK1 and between HER2 and SK1 was obtained using MDA-MB-453 cells. RESULTS: High S1P(4) expression is associated with shorter disease-free (P=0.014) and disease-specific survival (P=0.004), and was independent on multivariate analysis. In addition, patients with tumours that contain high and low levels of SK1 and S1P(4), respectively, have a significantly shorter disease-free survival (P=0.043) and disease-specific survival (P=0.033) compared with patients whose tumours contain both low S1P(4) and SK1 levels. In addition, high tumour expression of SK1 was significantly associated with shorter disease-specific survival (P=0.0001) in patients with HER2-positive tumours. Treatment of MDA-MB-453 cells with the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) reduced the basal and S1P/S1P(4)-induced activation of ERK-1/2 and altered HER2 trafficking in these cells. CONCLUSION: These findings highlight an important role for S1P(4) and SK1 in ER(-) breast cancer progression.

Therapeutic potential of targeting sphingosine kinases and sphingosine 1-phosphate in hematological malignancies.

Sphingolipids, such as ceramide, sphingosine and sphingosine 1-phosphate (S1P) are bioactive molecules that have important functions in a variety of cellular processes, which include proliferation, survival, differentiation and cellular responses to stress. Sphingolipids have a major impact on the determination of cell fate by contributing to either cell survival or death. Although ceramide and sphingosine are usually considered to induce cell death, S1P promotes survival of cells. Sphingosine kinases (SPHKs) are the enzymes that catalyze the conversion of sphingosine to S1P. There are two isoforms, SPHK1 and SPHK2, which are encoded by different genes. SPHK1 has recently been implicated in contributing to cell transformation, tumor angiogenesis and metastatic spread, as well as cancer cell multidrug-resistance. More recent findings suggest that SPHK2 also has a role in cancer progression. This review is an overview of our understanding of the role of SPHKs and S1P in hematopoietic malignancies and provides information on the current status of SPHK inhibitors with respect to their therapeutic potential in the treatment of hematological cancers.

Short-term local delivery of an inhibitor of Ras farnesyltransferase prevents neointima formation in vivo after porcine coronary balloon angioplasty.

BACKGROUND: Mitogenic stimuli present at the site of coronary arterial balloon injury contribute to the progression and development of a restenotic lesion, many signaling through a common pathway involving the small G protein p21(ras). Our aim was to demonstrate in biochemical studies that farnesyl protein transferase inhibitor III (FPTIII) is an inhibitor of p21(ras) processing and that when it is given locally in vivo at the site of coronary balloon injury in a porcine model, it can inhibit neointima formation. METHODS AND RESULTS: FPTIII (1 to 25 micromol/L) concentration-dependently reduced p21(ras) levels in porcine coronary artery smooth muscle cell membranes. FPTIII also prevented p42/p44 MAPK activation and DNA synthesis in response to platelet-derived growth factor in these cells at a concentration of 25 micromol/L. Application of 25 micromol/L FPTIII locally for 15 minutes to balloon-injured porcine coronary arteries in vivo prevented neointima formation assessed at 4 weeks, reduced proteoglycan deposition, and inhibited adventitial hypertrophy. Coronary arteries from FPTIII-treated pigs had no deterioration in contraction or in endothelium-dependent relaxation. CONCLUSIONS: The study demonstrates in the pig that short-term local delivery of inhibitors of p21(ras)-dependent mitogenic signal transduction prevents restenosis after balloon angioplasty.

The inhibition of adenylyl cyclase activity in isolated lung membranes by muscarinic and alpha-adrenoceptor agonists: role of G-protein alpha and beta gamma sub-units.

Forskolin (10 microM) failed to abolish the GppNHp- (0.1 nM) dependent inhibition of adenylyl cyclase activity in isolated membranes. Whilst clonidine (1 microM), an alpha 2-adrenoceptor agonist, inhibited adenylyl cyclase activity it did not protentiate the GppNHp-dependent inhibition. This indicates that low concentrations of the guanine-nucleotide activate sufficient Gi to inhibit adenylyl cyclase activity maximally and that clonidine inhibits this enzyme via a similar route. These data support a role of alpha i sub-unit inhibition of adenylyl cyclase activity. In contrast, forskolin (10 microM) abolished the GppNHp (0.001-0.1 nM) dependent inhibition of adenylyl cyclase activity in membranes where adenylyl cyclase activity is limited, i.e. where activity has been depleted by approximately 80%. In this case, inhibition of adenylyl cyclase by beta gamma sub-units is implicated and only becomes evident under these conditions. Adenylyl cyclase is also inhibited by muscarinic receptor agonist, methacholine and by the alpha 2-adrenoceptor agonists, clonidine and nor-adrenaline. Both classes of agonist also elicit an increase in the cholera toxin-catalysed ADP-ribosylation of the splice variant forms of Gs alpha and of a polypeptide of 41,000 M(r). The ADP-ribosylation of the 41,000 M(r) polypeptide is inhibited by GTP (100 microM) and therefore displays characteristics similar to Gi alpha. Muscarinic receptor and alpha 2-adrenoceptor agonists appear to inhibit adenylyl cyclase activity in lung membranes predominantly via Gi alpha. Lung expresses both type II and IV adenylyl cyclase which are stimulated by direct interaction with beta gamma sub-units and this is conditional upon the co-incident activation of Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived growth factor activates a mammalian Ste20 coupled mitogen-activated protein kinase in airway smooth muscle.

We have investigated the mechanisms regulating p38MAPK in airway smooth muscle cells. Incubation of cells with platelet-derived growth factor (PDGF) stimulated MAPKA kinase-2 activity, a kinase immediately down-stream of P38MAPK. Preincubation of the cells with forskolin (10 microM), which stimulated a 20-fold increase in intracellular cAMP formation, inhibited this response. An antibody raised against subdomain VI of yeast Ste20 kinase co-immunoprecipitated p38MAPK from cell lysates. The immunoprecipitated kinase(s) was shown to catalyse the phosphorylation of myelin basic protein (MBP) and to activate purified MAPKAP kinase-2. Incubation of cells with PDGF did not increase the amount of p38MAPK isolated in the anti-Ste20 immunoprecipitate. However, the kinase phosphorylated MBP and stimulated purified MAPKAP kinase-2 activity more effectively than kinase from control cells. The preincubation of cells with forskolin (10 microM) reduced the amount of P38MAPK in the immunoprecipitate and this correlated with a decrease in kinase activity. We conclude the PDGF induces the activation of p38MAPK, whereas forskolin elicits its dissociation from the complex with Ste20. Therefore, Ste20/p38MAPK may form part of a signal transduction pathway linked to activation of MAPKAP kinase-2 in ASM cells.

Bradykinin-stimulated phosphatidate and 1,2-diacylglycerol accumulation in guinea-pig airway smooth muscle: evidence for regulation 'down-stream' of phospholipases.

Bradykinin-treatment of cultured airway smooth muscle (ASM) induced the formation of [3H]1,2-diacylglycerol ([3H]1,2-DG), [3H]1,3-diacylglycerol ([3H]1,3-DG) and [3H]phosphatidic acid ([3H]PtdOH) in [3H]palmitate-labelled cells and of [3H]choline in [3H]methyl choline-labelled cells. [3H]1,2-DG and [3H]1,3-DG responses were biphasic with an initial transient phase from 0-2 min and a second sustained phase to 10 min. In contrast, [3H]PtdOH accumulation plateaued at 2 min stimulation as did [3H]choline formation. The bradykinin-stimulated [3H]1,2-DG and [3H]PtdOH responses exhibited similar concentration dependencies (EC50 values: [3H]1,2-DG 5.14 +/- 2.82 nM; [3H]1,3-DG 4.95 +/- 1.12 nM; [3H]PtdOH 1.52 +/- 0.82 nM). In contrast, PMA elicited a [3H]PtdOH response, but was without effect upon [3H]DG levels. Bradykinin-induced accumulation of [3H]1,2-DG and [3H]PtdOH was insensitive to blockade by a bradykinin B2-receptor antagonist, NPC567 (40 microM) and the B1-receptor agonist, Des-Arg9-bradykinin, (10 microM) failed to elicit a response. These observations are similar to those obtained previously for bradykinin-stimulated phospholipase D activity in ASM (Pyne S. and Pyne N. J., Br. J. Pharmac. 110, 477-481, 1993). Thus, both bradykinin-stimulated 1,2-DG and PtdOH accumulation may also be regulated via a novel B3-receptor. Bradykinin-stimulated formation of [3H]PtdOH was partially inhibited by butan-1-ol (by 47.25 +/- 12.7%, n = 3) which had no effect upon basal or bradykinin-stimulated levels of [3H]1,2-DG or upon basal [3H]PtdOH.(ABSTRACT TRUNCATED AT 250 WORDS)

PDGF-stimulated cyclic AMP formation in airway smooth muscle: assessment of the roles of MAP kinase, cytosolic phospholipase A2, and arachidonate metabolites.

Platelet-derived growth factor (PDGF) stimulates cyclic AMP (cAMP) synthesis in cultured guinea-pig airway smooth muscle (ASM) cells. However, this stimulation is normally countered by the action of cAMP phosphodiesterases. Thus, cAMP synthesis was observed only in cells pre-treated with either 3-isobutyl-1-methylxanthine (IBMX) or with cholera toxin. cAMP synthesis was inhibited by pre-treating cells with well-defined inhibitors of arachidonate metabolite synthesis, such as AACOCF3 [a cytosolic phospholipase A2 (cPLA2) inhibitor] and indomethacin (a cyclooxygenase inhibitor). This suggests that arachidonate metabolites (e.g., prostaglandins) released in response to PDGF stimulate cAMP synthesis. The presence of functional prostaglandin (PG) receptors was confirmed by experiments that showed that exogenous PGE2 stimulated cAMP formation. cPLA2 is regulated by mitogen-activated protein kinase (MAPK) in a number of cell types. The presence of this pathway in ASM cells and its role in regulating arachidonate metabolism were supported by the finding that pre-treatment of cells with PD098059 (an inhibitor of mitogen-activated protein kinase kinase-1 activation) reduced PDGF-stimulated cAMP synthesis. The cAMP formed in response to the arachidonate metabolites subsequently reduced the PDGF-dependent activation of c-Raf, MAPK, and DNA synthesis, suggesting the presence of a negative feedback pathway.

Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells.

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.

The role of the cyclic GMP-inhibited cyclic AMP-specific phosphodiesterase (PDE3) in regulating clonal BRIN-BD11 insulin secreting cell survival.

We report here that the cyclic GMP-inhibited cyclic AMP specific phosphodiesterase (PDE3B) is expressed as a membrane-bound protein in clonal insulin-secreting BRIN-BD11 cells. This was shown using SKF94836 (PDE3 inhibitor) which maximally inhibited membrane-bound cyclic AMP PDE activity by approximately 25-30% and by RT-PCR. We also demonstrated that insulin growth factor-1 (IGF-1) activates PDE3B in BRIN-BD11 cells. We therefore evaluated the role of phosphoinositide 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathways in regulating this enzyme. We report here that the PI3K inhibitor, wortmannin, prevented the IGF-1-dependent stimulation of PDE3B activity. In contrast, the inhibitor of MEK-1 activation, PD098059 (which reduced IGF-1-stimulated p42/p44 MAPK phosphorylation), had no effect on PDE3B activation. Furthermore, IGF-1-dependent stimulation of p42/p44 MAPK and PDE3B was abolished in serum-deprived cells and this was associated with apoptosis. We propose that the deregulation of the PI3K/PDE3B pathway might result in increased intracellular cyclic AMP accumulation, which promotes apoptosis. This was supported by the finding that the adenylyl cyclase activator, forskolin, also induced apoptosis. Finally, we found that orthovanadate (a phosphotyrosine phosphatase inhibitor) fully restored the activation of p42/p44 MAPK in serum-deprived cells, but had only a small effect on PDE activity. This confirmed that p42/p44 MAPK is on a separate pathway to PDE3B. Therefore, IGF-1-dependent regulation of PDE3B may be linked to cell survival through PI3K and not p42/p44 MAPK.

The gamma subunit of the rod photoreceptor cGMP phosphodiesterase can modulate the proteolysis of two cGMP binding cGMP-specific phosphodiesterases (PDE6 and PDE5) by caspase-3.

We have investigated whether the proteolysis of members of the cGMP binding phosphodiesterases (PDE6, PDE5A1, and PDE10A2) by caspase-3 is modulated by the gamma inhibitor subunit of PDE6. We show here that purified caspase-3 proteolyses PDE6, an enzyme composed of two nonidentical catalytic subunits (termed alpha and beta) with molecular mass of 88 and 84 kDa. The proteolysis of PDE6 produced a single fragment with a molecular mass of 78 kDa. This corresponds to the possible cleavage of the caspase-3 consensus DFVD site (amino acids: 164-168) in the alpha subunit and leads to a 50% decrease in the cGMP hydrolysing activity of the enzyme. The addition of rod PDEgamma to the incubation completely blocked the cleavage of PDE6 by caspase-3. In contrast, rod PDEgamma converted PDE5A1 (molecular mass of 98 kDa) to a better substrate for caspase-3. This resulted in the formation of four major fragments with molecular mass of 82-83, 67, 43, and 34 kDa. In addition, caspase-3 induced an approximately 80% reduction in the activity of a partially purified preparation of PDE5A1 in the presence of rod PDEgamma. Caspase-3 also cleaved PDE10A2 (molecular mass of 95 kDa) to a single 48-kDa fragment. This was consistent with cleavage of the DLFD site (amino acids: 312-315) in PDE10A2. In contrast with both PDE6 and PDE5A1, rod PDEgamma was without effect on this enzyme. These data show that rod PDEgamma interacts with at least two members of the cGMP binding PDE family (PDE5A1 and PDE6) and can exert differential effects on the cleavage of these enzymes by caspase-3.

The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes.

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.

Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi.

The antiserum AS7 can specifically immunoprecipitate alpha-Gi from membrane extracts as well as from a mixture of purified alpha-Gi and alpha-Go as ascertained using [32P]ADP-ribosylated G-proteins. Using this antiserum to immunoprecipitate alpha-Gi from hepatocytes labelled with 32P it was evident that alpha-Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of alpha-Gi. This was accompanied by the loss of inhibitory effect of Gi on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH-glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD+-dependent, [32P]ADP-ribosylation of alpha-Gi in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of alpha-Gi in intact hepatocytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves alpha-Gi in its holomeric state.

Differential effects of B2 receptor antagonists upon bradykinin-stimulated phospholipase C and D in guinea-pig cultured tracheal smooth muscle.

1. Guinea-pig tracheal smooth muscle cells were isolated and maintained in culture for 14-21 days prior to the study of the effect of a selective bradykinin B1 agonist and B2 antagonists upon bradykinin-stimulated phospholipase C and D activities. 2. Bradykinin-stimulated phospholipase C activity was determined by mass measurement of inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) in unlabelled cells, whereas phospholipase D activity was assayed by the accumulation of [3H]-phosphatidylbutanol ([3H]-PtdBut) in [3H]-palmitate-labelled cells, which were stimulated in the presence of butan-1-o1 (0.3%, v/v). 3. Bradykinin elicited the rapid and transient formation of Ins(1,4,5)P3, in a concentration-dependent manner (log EC50 = -7.55 +/- 0.1 M, N = 3). Bradykinin also rapidly activated the concentration-dependent (log EC50 = -8.3 +/- 0.4 M, n = 3) phospholipase D-catalysed accumulation of [3H]-PtdBut; the accumulation of [3H]-PtdBut was sustained. These effects were not inhibited by pretreatment of the cells with indomethacin (1 microM). 4. The bradykinin B1 agonist, desArg9-bradykinin (1 microM) was without effect upon phospholipase C or phospholipase D activity. Bradykinin-stimulated (10 nM, EC40) Ins(1,4,5)P3 formation was inhibited by B2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-bradykinin (NPC 567) and D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (NPC 349), with log IC50 values of -6.3 +/- 0.5 M and -6.3 +/- 0.4 M, respectively. However, bradykinin-stimulated (10 nM, EC100) [3H]-PtdBut accumulation was poorly inhibited and with low potency by each B2 receptor antagonist and bradykinin-stimulated phospholipase D activity persisted at concentrations of antagonist that completely blocked bradykinin-stimulated Ins(1,4,5)P3 formation (30 microM). 5. These observations suggest that the activation of phospholipase C by bradykinin may be mediated through a bradykinin B2 receptor population, whereas bradykinin-stimulated phospholipase D may be activated via a distinct population of bradykinin receptors that do not appear to be either B1 or B2 receptor types, based upon pharmacological specificity. The mechanism of the activation of phospholipase D by bradykinin and the role of the putative B3 bradykinin receptor are discussed.

The identification of apparently novel cyclic AMP and cyclic GMP phosphodiesterase activities in guinea-pig tracheal smooth muscle.

Phosphodiesterase (PDE) activities that were capable of hydrolysing cyclic AMP (Km = 6.8 +/- 2 microM) and cyclic GMP (Km = 6.7 +/- 1.6 microM) were isolated from tracheal smooth muscle. These enzyme(s) activities were insensitive to stimulation by calcium/calmodulin and to inhibition by cyclic GMP, rolipram (type IV inhibitor) and siguazodan (type III inhibitor). Zaprinast was a relatively poor inhibitor of both cyclic AMP and cyclic GMP hydrolysis (IC50 = 46 +/- 9 microM and 45 +/- 14 microM respectively). These results suggest that tracheal smooth muscle may contain an apparently novel PDE. However, KCl (30 mM) which facilitates calcium entry in cells, depressed bradykinin-stimulated intracellular cyclic AMP formation, suggesting that the type I PDE may be functionally present. We suggest that considerable caution be exercised in identifying apparently novel PDE isoforms.

Effects of type-selective phosphodiesterase inhibitors on glucose-induced insulin secretion and islet phosphodiesterase activity.

1. We examined various type-selective phosphodiesterase (PDE) inhibitors on glucose-induced insulin secretion from rat isolated islets, on islet PDE activity and on islet cyclic AMP accumulation in order to assess the relationship between type-selective PDE inhibition and modification of insulin release. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 10(-5)-10(-3) M), as well as the type III selective PDE inhibitors SK&F 94836 (10(-5)-10(-3) M), Org 9935 (10(-7)-10(-4) M), SK&F 94120 (10(-5)-10(-4) M) and ICI 118233 (10(-6)-10(-4) M) each caused concentration-dependent augmentation (up to 40% increase) of insulin release in the presence of a stimulatory glucose concentration (10 mM), but not in the presence of 3 mM glucose. 3. Neither the type IV PDE inhibitor rolipram (10(-4) M) nor the type I and type V PDE inhibitor, zaprinast (10(-4)-10(-3) M) modified glucose-induced insulin release when incubated with islets, although a higher concentration of rolipram (10(-3) M) inhibited secretion by 55%. However, when islets were preincubated with these drugs followed by incubation in their continued presence, zaprinast (10(-6)-10(-4) M) produced a concentration-dependent inhibition (up to 45% at 10(-4) M). Under these conditions, rolipram inhibited insulin secretion at a lower concentration (10(-4) M) than when simply incubated with islets. 4. A combination of SK&F 94836 (10(-5) M) and forskolin (5 x 10(-8) M) significantly augmented glucose-induced insulin secretion (30% increase), although neither drug alone, in these concentrations, produced any significant effect. 5. Islet cyclic AMP levels, which were not modified by forskolin (10-6 M), SK&F 94836 (10-4 M) or Org 9935 (10-5 M) were significantly elevated (approximately 3.7 fold increase) by forskolin inc ombination with either SK&F 94836 or Org 9935.6 Homogenates of rat islets showed a low Km (1.7 microM) and high Km (13 microM) cyclic AMP PDE in the supernatant fractions (from 48,000 g centrifugation), whereas the particulate fraction showed only a low Km (1.4 microM) cyclic AMP PDE activity.7. The PDE activity of both supernatant and pellet fractions were consistently inhibited by SK&F94836 or Org 9935, the concentrations required to reduce particulate PDE activity by 50% being 5.5 and 0.05 microM respectively.8 Rolipram (10-5 10-4 M) did not consistently inhibit PDE activity in homogenates of rat islets and zaprinast (10-4 M) consistently inhibited activity by 30% in the supernatant fraction, but not consistently in the pellet.9 These data are consistent with the presence of a type III PDE in rat islets of Langerhans.

Assessment of agonism at G-protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells.

Several different molecular species of phosphatidic acid (PA) bind to a G-protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from G(i/o)). The de-acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an approximately 100 - 1000 fold lower potency compared with the different molecular species of PA. RT - PCR using gene-specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.

Muscarinic blockade of beta-adrenoceptor-stimulated adenylyl cyclase: the role of stimulatory and inhibitory guanine-nucleotide binding regulatory proteins (Gs and Gi).

1. The functional antagonism that exists between muscarinic and beta-adrenoceptor function in guinea-pig tracheal smooth muscle was investigated by assessing Gs and Gi regulated adenylyl cyclase activity in isolated membranes. 2. Membranes from guinea-pig tracheal smooth muscle contain both Gi alpha and Gs alpha as assessed by Western blots with anti-G-protein antibodies. 3. GppNHp, a non-hydrolysable analogue of guanosine 5'-triphosphate (GTP), was shown to stimulate adenylyl cyclase activity at high concentrations (10(-6)-10(-4) M). GppNHp also produced a concentration-dependent reduction in pertussis toxin-catalysed adenosine diphosphate (ADP)-ribosylation of Gi alpha. 4. Pretreatment of tracheal smooth muscle slices with methacholine (10(-6) M) provoked a blockade of isoprenaline plus GTP, GppNHp- and GTP-stimulated adenylyl cyclase. 5. Addition of methacholine to membranes did not trigger inhibition of GTP-stimulated adenylyl cyclase activity but did block the isoprenaline-mediated augmentation of GTP-stimulated adenylyl cyclase activity. 6. Pretreatment of tracheal smooth muscle with methacholine (10(-6) M) provoked a blockade of cholera toxin-catalysed NAD(+)-dependent ADP-ribosylation of Gs alpha. 7. Phorbol-12-myristate 13-acetate (PMA)-treatment of tracheal smooth muscle slices actually enhanced GppNHp-stimulated adenylyl cyclase activity in subsequently prepared membranes. 8. We suggest that methacholine in addition to inhibiting adenylyl cyclase via a Gi-dependent mechanism induces a functional inactivation of Gs activity. These results together may explain the functional antagonism that exists between increased muscarinic tone and the ability of beta-adrenoceptor agonists to provoke excitation-contraction uncoupling.

The identification of DL-threo dihydrosphingosine and sphingosine as novel inhibitors of extracellular signal-regulated kinase signalling in airway smooth muscle.

We present entirely novel evidence that DL-threo dihydrosphingosine and sphingosine are inhibitors of the extracellular signal-regulated kinase (ERK) signalling cassette in mammalian cells. We show that DL-threo dihydrosphingosine is effective against both growth factor- and G-protein-dependent activation of ERK. We conclude that DL-threo dihydrosphingosine may represent an important pharmacological cell-permeable agent that may be usefully employed to block smooth muscle cell proliferation.

Effect of type-selective inhibitors on cyclic nucleotide phosphodiesterase activity and insulin secretion in the clonal insulin secreting cell line BRIN-BD11.

1. The cyclic nucleotide phosphodiesterases (PDEs) present in an insulin secreting cell line, BRIN - BD11, were characterized using calcium/calmodulin, IGF-1, isoenzyme-selective PDE inhibitors and RT - PCR. 2. Calmodulin activated cyclic AMP or cyclic GMP PDE activity in pellet and was 3 fold (P=0.002) more potent in activating cyclic nucleotide hydrolysis in pellet compared with supernatant fractions. 3. The PDE1/PDE5 inhibitor zaprinast inhibited both cyclic AMP and cyclic GMP PDE activity in both pellet and supernatant fractions of cell homogenates by a maximum of around 25% (IC(50) 1 - 5 microM), while rolipram (PDE4 selective) inhibited only cyclic AMP hydrolysis. 4. The PDE3-selective inhibitors Org 9935 (0.02 - 10 microM) and siguazodan (0.1 - 10 microM) inhibited cyclic AMP PDE activity in the pellet but not the supernatant fractions of cell homogenates, with a maximum inhibition of about 30%. IGF-1 (2 - 7.5 ng ml(-1)) potently augmented this PDE activity. 5. RT - PCR using specific primers for PDE3B, but not for PDE3A, amplified, from BRIN - BD11 cell total RNA, a 351 base pair product that was >97% homologous with rat adipose tissue PDE3B. 6. IBMX, Org 9935, siguazodan and rolipram (1 - 50 microM), but not zaprinast, each augmented glucose-induced insulin secretion in the presence of 16.7 mM but not 1 mM glucose. 7. These findings, in a clonal insulin secreting cell line, are consistent with an important role for PDE3B in regulating the pool of cyclic AMP relevant to the modulation of glucose-induced insulin secretion.