Antithrombotic activity of BMY-43351, a new imidazoquinoline with enhanced aqueous solubility

BMY-43351 is a new broad-spectrum inhibitor of platelet aggregation with greater aqueous solubility than earlier analogs from the imidazoquinoline series. This report compares the antithrombotic activity of BMY-43351 to that of two other imidazoquinolines: BMY-20844, a simply-substituted compound, and BMY-21638, a more potent ether-linked side chain analog. All of these compounds act, at least in part, via inhibition of platelet low-Km cyclic AMP phosphodiesterase. Antithrombotic activity was assessed in the rabbit ear chamber-biolaser preparation, an animal model of small vessel thrombosis, and in the canine coronary artery stenosis-occlusion model of large vessel thrombosis. BMY-43351 was found to be remarkably potent in the biolaser model, with an EDso of 0.074 mg/kg p.o. In comparison, compounds such as aspirin, ticlopidine, sulfinpyrazone, and dipyridamole demonstrate little or no activity at much higher doses, (eg. 100 mg/kg p.o.). Other inhibitors of platelet low Km cyclic AMP phosphodiesterase are active but substantially weaker than BMY-43351. Similarly, in the coronary artery stenosis-occlusion model, BMY-43351 demonstrated impressive activity, significantly inhibiting arterial thrombosis at intraduodenal doses as low as 1 micrograms/kg. The potential use of BMY-43351 as adjunct therapy in thrombolysis was suggested in a series of experiments where this drug was used in combination with a thrombolytic regimen of stretokinase plus heparin. In this experimental setting, time to reperfusion was reduced from 42 +/- 5 minutes to 11 +/- 5 minutes, and reocclusion was totally inhibited.     

Effect of cAMP phosphodiesterase inhibitors on ADP-induced shape change, cAMP and nucleoside diphosphokinase activity of rabbit platelets

The effects of cAMP phosphodiesterase inhibitors on ADP-induced shape change and cAMP concentrations have been studied. Caffeine (10 mM), theophylline (8 mM), dipyridamole (0.2 mM), or papaverine (0,05 mM) prevented the shape change of washed rabbit platelets induced by 0.4 microM ADP. At these concentrations, none of these cAMP phosphodiesterase inhibitors increased 14C-cAMP in platelets in which the cytoplasmic adenine nucleotides had been labelled with 14C-adenine. By a protein binding assay, only papaverine by itself increased platelet cAMP above its basal level. These results indicate that two pools of cAMP may exist in platelets. Both methods showed that stimulation of platelet adenylate cyclase with PGE1 (1 microM) resulted in an increase in platelet cAMP and all these cAMP phosphodiesterase inhibitors potentiated this increase caused by PGE1. By themselves, some of these compounds may act through mechanisms that do not involve platelet cAMP. The effects of these cAMP phosphodiesterase inhibitors on platelet nucleoside diphosphokinase (NDK) activity were also investigated. At concentrations that prevented ADP-induced shape change, papaverine and dipyridamole had no effect on the formation of 14C-ATP from 14C-ADP by washed rabbit platelets. The methylxanthines partially inhibited NDK activity of washed rabbit platelets and of isolated platelet membranes, probably due to the structural similarity between the adenine ring of ADP and these substances. However, adenine (8 mM) inhibited ADP-induced shape change and platelet NDK activity but was a less effective inhibitor of ADP-induced platelet aggregation. Thus it seems unlikely that interference with platelet NDK or the ADP receptor is the major mechanism by which the methylxanthines inhibit platelet functions.     

Different effects of cAMP and cGMP derivatives on the activity of an identified neuron: Biochemical and electrophysiological analysis

Eight-position substituted cAMP and cGMP derivatives, and phosphodiesterase inhibitors, modify endogenous 'bursting' activity in Aplysia neuron R15. Several different patterns of activity were elicited depending on the agent used. 8-Benzylthio-cAMP or 8-parachlorophenylthio-cAMP, at concentrations between 5 muM and 0.3 mM, markedly enhanced the depth and duration of the interburst hyperpolarization, and in some cells bursting was inhibited completely. In contrast, 8-parachlorophenyl-thio-cGMP treatment led to some depolarization and to the appearance of long slow bursts, with little effect on the interburst phase. When the parachlorophenylthio-derivatives of cAMP and cGMP were added together at equal concentrations, a pattern consisting of long bursts interrupted by long and deep interburst hyperpolarizations was observed. This pattern could also be elicited by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). IBMX inhibited cAMP and cGMP phosphodiesterases and caused both cAMP and cGMP to accumulate in intact ganglia and in individual identified neuronal cell bodies including that of R15. Another phosphodiesterase inhibitor, Ro 7-2956, was a more potent inhibitor of cAMP than of cGMP phosphodiesterase; Ro 7-2956 also modified bursting activity, and seemed to enhance preferentially the interburst hyperpolarization. At high concentrations the 8-substituted cAMP and cGMP derivatives also inhibited cAMP and cGMP phosphodiesterases. The 8-parachlorophenylthio-derivatives of cAMP and cGMP were indistinguishable from each other in this assay, and thus phosphodiesterase inhibition cannot be responsible for their differential effects on bursting activity. The derivatives stimulated protein kinase activity in Aplysia ganglion homogenates, as measured by the incorporation of 32P from ATP into histone. IBMX and Ro 7-2956 had no detectable effect on protein kinase activity. The concentrations of cAMP and cGMP derivatives required for protein kinase activation (10(-8)M-10(-6)M) were much lower than those required for phosphodiesterase inhibition (10(-5)M-10(-3)M). Thus, differential protein phosphorylation is more likely to be responsible for the effects of cAMP and cGMP derivatives on neuron R15 bursting activity than is differential phosphodiesterase inhibition.     

Role of plasma adenosine in the antiplatelet action of HL 725, a potent inhibitor of cAMP phosphodiesterase: species differences

The potent inhibitor of platelet cAMP phosphodiesterase (PDE) HL 725 (9,10-Dimethoxy-2-mesitylimino-3-methyl-3, 4,6,7-tetrahydro-2H-pyrimido(6,1-A)-isoquinoline-4-one-hydrochloride), was examined for its effects on human and rat platelet aggregation. Strong inhibitory effects are seen on collagen-induced platelet aggregation both in rat platelet-rich plasma (PRP) (IC50, 54 +/- 12 nM) and whole blood (IC50, 57 +/- 25 nM). Compared to the effects on rat platelets, HL 725 is about two-fold less inhibitory in human PRP (IC50, 94 +/- 29 nM) and whole blood (IC50, 126 +/- 50 nM). The inhibitory action of HL 725 can be reversed by washing and resuspension of the platelets, suggesting that HL 725 does not bind tightly to cAMP PDE. If human or rat PRP is pretreated with adenosine deaminase, an enzyme that degrades adenosine or 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, the inhibitory effect of HL 725 is reversed. Similar blockade of the inhibitory actions of several other inhibitors of cAMP PDE such as RA 233, RX-RA 69 (analogs of dipyridamole) and oxagrelate is seen by adenosine deaminase pretreatment. The nucleoside transport inhibitors, dilazep and dipyridamole which are non-inhibitory alone to platelet aggregation, strongly potentiate (about 10-fold) the inhibitory action of HL 725 on collagen-induced platelet aggregation in human whole blood. However, if the whole blood is pretreated with adenosine deaminase, no inhibitory effect of dipyridamole plus HL 725 is seen on platelet aggregation. These studies demonstrate that plasma adenosine plays a crucial role in the antiaggregatory actions of HL 725 and several other inhibitors of cAMP PDE both in human and rat blood.     

Potentiation of the antiplatelet action of adenosine in whole blood by dipyridamole or dilazep and the cAMP phosphodiesterase inhibitor, RA 233

Adenosine (Ado, 10-50 microM), a potent inhibitor of ADP-induced human platelet aggregation in platelet-rich plasma (PRP), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) which are resistant to deamination (2-fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibit aggregation in whole blood with IC50 values of 12 microM, 2.3 microM and 0.26 microM, respectively. The inhibitory effect of NECA (200 nM) is potentiated by the platelet cAMP phosphodiesterase (PDE) inhibitor RA 233 (5 microM). Inhibition of the erythrocytic nucleoside transport system by dilazep (1 microM) or dipyridamole (10 microM), or blockade of Ado metabolism by 2'-deoxycoformycin (5 microM) plus 5-iodotubercidin (10 microM), evokes the antiaggregatory action of Ado in whole blood (IC50 congruent to 2 microM). RA 233 (5 microM) potentiates Ado-mediated inhibition about 10-fold when nucleoside transport or Ado metabolism is blocked. Ado (10 microM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleoside transport is inhibited by dilazep or dipyridamole, or when Ado metabolism is blocked by 2'-deoxycoformycin and 5-iodotubercidin, 50-60% of the Ado remains in the plasma after 5 min. These results show that the failure of Ado to inhibit platelet aggregation in whole blood results from its rapid uptake and metabolism by erythrocytes. More importantly, these data emphasize the key role of nucleoside transport inhibition in the antiplatelet actions of dipyridamole and dilazep. In addition, superior therapeutic results may be obtained from the combination of blockade of nucleoside transport system with inhibition of platelet cAMP PDE.     

Regulation of cAMP levels by protein kinase C in C6 rat glioma cells

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13-dibutyrate (PDbU). An IC50 for PDbU of 38 +/- 5 nM and 62 +/- 8 nM was observed in the isoproterenol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproterenol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181-186, 1987). An IC50 for phospholipase C of 6.0 +/- 0.1 x 10(-1) and 7.0 +/- 0.1 x 10(-1) units/ml was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU-mediated inhibition to beta-adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta-adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed.     

Differential regulation of P-selectin expression by protein kinase A and protein kinase G in thrombin-stimulated human platelets

P-selectin is rapidly translocated from platelet alpha-granules following activation. Intracellular cyclic AMP (cAMP) is a potent inhibitory pathway that results in global downregulation of platelet activation. While cAMP-dependent protein kinase (PKA) has long been considered as the main mediator of cAMP-dependent effects, no study has yet evaluated its effect on P-selectin expression in human platelets. Pretreatment of thrombin-stimulated platelets with forskolin resulted in a concentration- dependent inhibition of P-selectin expression that correlated with adenylyl cyclase activity. Inhibition of PKA with H-89 reversed cAMP-induced inhibition of P-selectin while cGMP-dependent protein kinase (PKG) inhibition with KT5823 significantly potentiated cAMP-dependent inhibition of P-selectin. Similar results were also observed in a platelet/neutrophil binding assay. In conclusion, cAMP-induced inhibition of P-selectin expression is, in large part, mediated through activation of PKA. PKG appears to be solicited for P-selectin expression when cAMP levels are elevated which suggest a cAMP/PKG-dependent pathway of platelet activation.     

Pharmacology of a potent, new antithrombotic agent, 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844)

The effects of 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844) on platelet function and experimental thrombosis were evaluated in a series of in vitro, ex vivo and in vivo experiments. The compound inhibited platelet aggregation in vitro in platelet rich plasma obtained from humans, rats and rabbits with EC50s of less than 1 microgram/ml when aggregation was induced by ADP, collagen or thrombin. Supra-additive interaction against ADP aggregation was also observed when BMY-20844 was combined with prostacyclin. BMY-20844 was orally active with an ex vivo ED50 in the rat of 3.2 mg/kg vs ADP. Significant antithrombotic activity was observed in two animal models (laser induced thrombosis in the microcirculation of the rabbit ear and coronary artery thrombosis in the dog). Inhibitions of 52% at 3 mg/kg p.o. in the laser model and 100% at 1 mg/kg i.d. in the coronary artery thrombosis model were obtained. Modest inotropic and hemodynamic effects were observed in ferrets and dogs. BMY-20844 was found to be a potent, specific inhibitor of platelet low Km cyclic AMP phosphodiesterase.     

The lack of correlation between inhibition of aggregation and cAMP levels with canine platelets

Arachidonic acid (AA) induced aggregation of canine platelets can be inhibited by various phosphodiesterase inhibitors (PDIs) with the order of potency IBMX greater than or equal to papaverine greater than Ro 20-1724 greater than theophylline. With aggregation induced by AA plus epinephrine (EPI), only IBMX and papaverine inhibited at 100 microM. None of these PDIs affected the basal cAMP levels but all potentiated the PGE1-stimulated cAMP production, with the order of potency being Ro 20-1724 greater than papaverine greater than IBMX greater than theophylline. PGE1 at 1 microM caused a sharp increase in cAMP and complete inhibition of platelet aggregation induced by AA plus EPI. However, when EPI was added before PGE1, there was no elevation of cAMP yet inhibition of aggregation still occurred. Our results indicated that inhibition of platelet aggregation does not require a measurable increase in cAMP.     

Cyclic AMP-mediated modulation of epileptiform afterdischarge generation in rat hippocampal slices

This study assessed the effects of drugs which manipulate the cAMP system on afterdischarges (ADs) induced in the CA1 region of rat hippocampal slices. The adenylate cyclase activator forskolin (50 microM) and the phosphodiesterase inhibitor rolipram (0.1 and 1 microM) enhanced AD generation. These effects were reversed by the cAMP-dependent protein kinase inhibitors H-89 (5 microM) and Rp-cAMPS (100 microM). These findings suggest that AD generation can be modulated through cAMP generation and the subsequent activation of the cAMP-dependent protein kinase.     

Distribution of cAMP and cAMP-dependent protein kinases in Aplysia sensory neurons

Sensitization of the gill- and siphon-withdrawal reflex in Aplysia is considered a simple form of learning. Previous work has provided physiological and pharmacological evidence that cAMP-dependent protein phosphorylation within identified sensory neurons of the abdominal ganglion underlies the short-term form of this behavioral modification. Our main goal in this paper is to determine the subcellular distribution of cAMP and to measure the amounts and properties of the 2 types of subunits (regulatory and catalytic) that constitute the cAMP-dependent protein kinase. Do these biochemical parameters differ in sensory cells from those in other parts of nervous tissue? We found that the increased cAMP synthesized under conditions of sensitization is distributed in 3 compartments in the neuron: most of it is free in the cytoplasm; the remainder is bound either to cytoplasmic or to particulate proteins, which are believed to be regulatory subunits of the cAMP-dependent protein kinase. Binding of cAMP within the neurons is a measure of activation of the kinase. At rest, 17% of the binding sites in sensory cells were occupied. After brief electrical stimulation of the connective, which released endogenous transmitter, occupancy increased to 34%. This treatment increased the amount of cAMP bound to the various binding proteins differentially. The biochemical characteristics of cAMP binding were found to be the same in sensory neurons as in the rest of the nervous system but different from those in muscle. Thus, memory and learning are likely to be mediated by enzymes that are shared by other nerve cells. We found that sensory neurons have greater cAMP-dependent protein kinase activity than other neurons, however, and as a result may be more sensitive to small increases of cAMP.     

Forskolin's effect on transient K current in nudibranch neurons is not reproduced by cAMP

Forskolin, a diterpene extracted from Coleus forskolii, stimulates the production of cAMP in a variety of cells and is potentially an important tool for studying the role of cAMP in the modulation of neuronal excitability. We studied the effects of forskolin on neurons of nudibranch molluscs and found that it caused characteristic, reversible changes in the amplitude and waveform of the transient K current, IA, and also activated an inward current similar to the cAMP-dependent inward current previously described in molluscan neurons. Forskolin altered the time course of IA activation and inactivation but did not affect the voltage dependence or the reversal potential of the current. IA normally inactivates exponentially, but in forskolin the time course of inactivation can be fit by the sum of 2 exponentials with an initial rate that is faster than the control and a final rate that is much slower. On depolarization in forskolin, IA begins to activate at the normal rate, but a slower component of activation is also seen. The changes in IA in the nudibranch cells were qualitatively different than the changes caused by forskolin in Aplysia bag cell neurons (Strong, 1984). Experiments were performed to determine whether these effects of forskolin require cAMP. Intracellular injection of cAMP, application of membrane-permeable analogs of cAMP, application of phosphodiesterase inhibitors, and intracellular injection of the active catalytic subunit of cAMP-dependent protein kinase did not affect the amplitude or waveform of IA. Also, the changes in IA that are caused by forskolin were not prevented or reversed by intracellular injection of an inhibitor of cAMP-dependent protein kinase. Cyclic AMP did, however, activate inward current at voltages near the resting potential. We conclude that the changes in IA and the activation of inward current represent separate affects of forskolin. The inward current appears to depend on an increase in intracellular cAMP, while the changes in IA do not. These experiments show that, in addition to activating adenylate cyclase, forskolin may have a separate direct affect on the transient K current.     

Pacemaker activity of locus coeruleus neurons: whole-cell recordings in brain slices show dependence on cAMP and protein kinase A

Noradrenergic neurons of the rat locus coeruleus (LC) are endogenous pacemakers that exhibit slow, tonic firing even in the complete absence of synaptic inputs. In the present study a time-dependent decline in LC spontaneous firing activity was found on intracellular dialysis during whole-cell recording with low-resistance patch electrodes; this decline was accentuated by a specific inhibitor of cAMP-dependent protein kinase (PKI5-24). Conversely, the inclusion of cAMP, 8-Br-cAMP, or the catalytic subunit of cAMP-dependent protein kinase (PKAcat) in the patch pipettes dose-dependently increased firing rate; intracellular PKI5-24 blocked both 8-Br-cAMP and PKAcat-induced firing in LC neurons. These results indicate that endogenous cAMP, via a phosphorylation-dependent route, drives tonic pacemaker activity in LC neurons.     

Effects of a thromboxane synthetase inhibitor and a cAMP phosphodiesterase inhibitor, singly and in combination, on platelet behaviour

The effects of dazoxiben, a thromboxane synthetase inhibitor, and AH-P 719, a cAMP phosphodiesterase inhibitor, on arachidonic acid (AA)-induced platelet behaviour were determined. The levels of cAMP present in platelet-rich plasma (PRP) after stimulating the platelets with AA in the absence and presence of the agents were also measured. AH-P 719, as well as dazoxiben, was more effective as an inhibitor of AA-induced platelet behaviour in PRP from some individuals than in PRP from others, and the effectiveness with which it inhibited platelet behaviour paralleled that of dazoxiben. A combination of both agents was more effective than either agent alone. Both AH-P 719 and dazoxiben increased the level of cAMP in AA-stimulated platelets but again they were more effective in PRP from some individuals than others. A combination of AH-P 719 and dazoxiben always resulted in higher levels of cAMP than either agent alone. These results imply that cAMP is involved in determining the effects of thromboxane synthetase inhibitors on platelet behaviour, and indicate that the anti-thrombotic potential of a combination of a thromboxane synthetase inhibitor and a cAMP phosphodiesterase inhibitor may be greater than that of the individual agents.     

Effect of chlorpromazine on the localization of cAMP phosphodiesterase

Summary Chlorpromazine (CPZ) at dosages of 10 mg/kg body weight (b. wt.) affected the cytochemical localization of cAMP-dependent phosphodiesterase (cAMP PDE) activity in the synapses of the rat frontal cortex. Postsynaptic cAMP PDE activity was inhibited, and presynaptic activity increased. CPZ also inhibited membrane-bound ATPase activity in the frontal cortex. The activity of Na⁺–K⁺-ATPase was significantly (P<0.005) inhibited in isolated plasma membranes from the rat frontal cortex. CPZ exposure also affected the cytochemical localization of cations with potassium pyroantimonate. Precipitate, which could be removed with 5 mm EGTA, was decreased in the mitochondria and synaptic vesicles in presynaptic areas after CPZ treatment. The incorporation of⁴⁵Ca²⁺ into slices of the rat frontal cortex was also significantly (P<0.001) inhibited by CPZ. This ultrastructural study shows that CPZ may affect biochemical events in an opposite manner in the pre- and post-synaptic areas of some neurons of the frontal cortex.     

Inhibition of rabbit platelet phosphodiesterase activity and aggregation by calcium channel blockers

Rabbit platelet cyclic AMP phosphodiesterase is inhibited by the three calcium channel blockers nifedipine, diltiazem, and verapamil with IC50's of 100 microM, 100 microM and 420 microM, respectively. Also, platelet aggregation induced by 4 microM ADP is inhibited by those compounds. Verapamil is the most potent aggregation inhibitor with an IC50 of 260 microM while diltiazem and nifedipine have IC50's of 630 microM and 840 microM, respectively. All three compounds display a maximum inhibition of 80-85%. Diltiazem and PGD2 potentiate the antiaggregatory activity of each other in that the inhibitions occurring in the presence of the combination of the two (at varying concentrations) exceed the calculated sums of the inhibitions produced by each alone. On the other hand, the antiaggregatory activities of verapamil or nifedipine, are additive with that of PGD2 in that no significant differences exist between the observed inhibitions produced by the combinations and the calculated summed values of the individual inhibitions. Our data suggest, therefore, that in addition to lowering intracellular calcium ions, which are required for platelet aggregation, the three calcium channel blockers inhibit the breakdown of cyclic AMP thereby promoting antiaggregation.     

Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.     

8-Chloro-cyclic adenosine monophosphate, a novel cyclic AMP analog that inhibits human glioma cell growth in concentrations that do not induce differentiation.

Cyclic AMP is supposed to play a role in cell growth and differentiation via activation of protein kinase A. The cAMP signal transduction pathway may therefore be used as a target for the development of anticancer drugs. We compared the effects of 8ClcAMP, a newly developed cAMP analog, to the effects of more commonly used cAMP analogs on the morphology and the proliferation of three human glioma cell lines. 8ClcAMP was the most potent growth inhibitor, exhibiting an IC50 of approximately 10 microM and inducing growth arrest in all three glioma cell lines at a concentration of 100 microM. The cAMP analogs 8CPTcAMP, dibutyryl cAMP, and 8BrcAMP were much less potent. If used in concentrations that induce growth arrest, both 8CPTcAMP and IBMX, but not 8ClcAMP, induced morphological differentiation of the glioma cells. Apparently, the growth-inhibiting effect of 8ClcAMP is not paralleled by its ability to induce morphological differentiation. The explanation for this phenomenon may be that 8ClcAMP does not exert its growth-inhibiting effect via activation of cAMP-dependent protein kinase. Two alternative mechanisms of action are discussed. Since 100 microM 8ClcAMP retarded the growth of normal rat astrocytes only to a marginal extent, without cytotoxic effects, it is concluded that 8ClcAMP may offer interesting perspectives in the treatment of malignant glioma.     

Nerve growth cones isolated from fetal rat brain. II. Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation

Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation were examined in growth cone particles (GCPs) prepared from fetal rat brain. Several major proteins which specifically bind a photoactivatable analogue of cAMP are observed in GCPs and correspond to isoelectric variants of the regulatory subunits of the cAMP-dependent protein kinase described in adult brain. We found no evidence for differential compartmentalization of specific cAMP-binding proteins in subcellular fractions of fetal brain or within GCPs. cAMP-stimulated phosphoproteins of GCPs are similar to cAMP-dependent protein kinase substrates characterized in nerve terminals (synaptosomes) of adult brain and include the nerve terminal-specific protein, synapsin I. However, as shown in the companion paper (Katz, F., L. Ellis, and K. H. Pfenninger (1985) J. Neurosci. 5: 1402-1411), this synaptic phosphoprotein is not the major kinase substrate in the GCP fraction. The finding of synapsin I in a subcellular fraction prepared from fetal brain suggests that components of the mature nerve terminal are already present in fetal brain during neuronal sprouting and prior to synaptogenesis.     

Cyclic AMP-mediated regulation of the resting membrane potential in myelin-forming oligodendrocytes in the isolated intact rat optic nerve

Myelin formation by oligodendrocytes has been shown to be regulated by cyclic AMP (cAMP) signaling pathways and to depend on the resting membrane potential (RMP). We therefore examined whether cAMP regulates the RMP of myelin-forming oligodendrocytes in isolated intact optic nerves of rats. Oligodendrocytes exhibited a significant developmental shift in the RMP from -37 mV at postnatal day (P)6-8 to -67 mV at P21-30. The regulation of RMP was examined further in myelin-forming oligodendrocytes in nerves aged P15-20. Raising intracellular cAMP with dbcAMP or forskolin induced a significant hyperpolarization in myelin-forming oligodendrocytes by 10-15 mV. Inhibition of cAMP-dependent protein kinase (PKA) with KT5720 depolarized the oligodendroglial RMP -30 mV, which was only partly reversed by dbcAMP. In contrast, inhibition of cAMP specific phosphodiesterase with rolipram had no significant effect on the oligodendroglial RMP or the cAMP-mediated hyperpolarization. Blockade of Kir with 100 microM BaCl(2) depolarized the oligodendrocyte RMP to -25 mV and inhibited the hyperpolarizing action of dbcAMP. The RMP was unaffected by agents that modulated ATP-sensitive potassium channels. The results provide evidence of a predominant role for Kir in setting the oligodendroglial RMP and show that cAMP regulates the oligodendroglial RMP, at least partly by a PKA-mediated pathway, possibly by modulating the activity of Kir.