Modification of GABAA receptor function by an analog of cyclic AMP

Chloride-dependent current responses to gamma-aminobutyric acid (GABA) were recorded from cultured rat hippocampal neurons under voltage clamp. In the presence of the membrane-permeable cyclic AMP analog, 8-bromo-cyclic AMP (8-Br-cAMP), the peak current response to GABA was reduced, although the reversal potential for the current evoked by GABA was unaltered; similar concentrations of 8-bromo-cyclic GMP did not alter the GABA response. 8-Br-cAMP also increased spontaneous activity of the neurons and blocked accommodation of firing. It is possible that the alterations in responses to GABA result from the activation of cyclic AMP-dependent protein kinase (cAMP-PK) and subsequent phosphorylation of the GABAA receptor.     

Resistance of afterhyperpolarizations in hippocampal pyramidal cells to prostaglandins and vasoactive intestinal polypeptide (VIP)

The afterhyperpolarization (AHP) that follows repetitive stimulation was recorded intracellularly from CA1 pyramidal neurons in the guinea pig hippocampal slice preparation. Although the late AHP could be blocked by histamine (1-10 microM), forskolin (10 microM) and 8-bromo-cyclic AMP (100 and 500 microM), neither prostaglandins D2, E1 and F2 alpha (0.5 microM) nor vasoactive intestinal polypeptide (0.5 microM) had any effect on the AHP, membrane potential, membrane resistance or action potential properties.     

The effect of forskolin on the response of acetylcholine receptors in frog sartorius endplate.

The effects of forskolin, a potent activator of adenylate cyclase, were examined on the frog neuromuscular junction. The depolarization elicited by ionophoretically applied acetylcholine was markedly reduced in amplitude and its time course was speeded up after treatment with 20-100 microM forskolin. The amplitude of extracellularly recorded miniature endplate potentials was decreased by the same factor as that of ionophoretically evoked responses and their decay time constant became shorter. All these changes, but not the shortening of spontaneous responses, were produced by 3-isobutyl-1-methylxantine and by N6-2'-O-dibutyryl cyclic AMP, both known to elevate intracellular cAMP. Forskolin-induced actions can be thus ascribed to the activation of cAMP-dependent protein kinase and to a direct effect on acetylcholine receptor channel.     

Autoradiographic localization of particulate cyclic AMP-dependent protein kinase in mammalian brain using [3H]cyclic AMP: implications for organization of second messenger systems

Cyclic AMP's regulatory role as an intracellular second messenger is well established. In brain and other tissues, specific proteins that bind cyclic AMP have been shown to be the regulatory subunits of cystolic and particulate cyclic AMP-dependent protein kinases. This study of the autoradiographic localization of specific [3H]cyclic AMP binding revealed the heterogeneous distribution of particulate cyclic AMP-dependent protein kinase in the mammalian central nervous system. Specific [3H]cyclic AMP binding to tissue sections was of high affinity (KD = 60 nM) and saturable (Bmax = 5 pmol/mg protein). Purine and pyrimidine nucleotide analogues demonstrated inhibition constants against [3H]cyclic AMP binding consistent with the specific labelling of cyclic AMP-dependent protein kinase (e.g. 8'-bromo-cyclic AMP: IC50 = 130 nM; inosine 3',5'-cyclic monophosphate: IC50 = 1 microM; uridine 3',5'-cyclic monophosphate: IC50 = 60 microM). Variations in the levels of [3H]cyclic AMP binding presumably reflect the presence of differing amounts of particulate cyclic AMP-dependent protein kinase in different neuronal populations. Highest densities were associated with neuronal cell layers such as the pyramidal cells of the piriform cortex and hippocampus, and granule cells of the dentate gyrus and cerebellum. High levels of binding were also found in other cortical and limbic structures, while moderate levels were found in hypothalamic, thalamic and midbrain areas. Excitotoxic lesions confirmed the localization of the enzyme in hippocampal pyramidal cells and cerebellar granule cells. Localizations reported in this study are largely consistent with results obtained using immunohistochemical methods to label cyclic AMP-dependent protein kinases. Recently, [3H]forskolin, a potent and selective activator of adenylate cyclase, the enzyme responsible for the formation of cyclic AMP from adenosine 5'-triphosphate, has been used to localize the activated catalytic component of this enzyme in rat brain. Regions described as being intensely labelled with [3H]forskolin (e.g. basal ganglia, hilus of the dentate gyrus and molecular layer of the cerebellum) were found to be associated with relatively low [3H]cyclic AMP binding levels. These findings suggest a marked difference between the localization of the two related enzyme entities. However, the distribution of the enzymes is indirectly correlated as high levels of particulate cyclic AMP-dependent protein kinase are present in the soma of neurons with high concentrations of adenylate cyclase in their terminals. Alternatively, it is possible that [3H]forskolin localizes only a subpopulation of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of either forskolin, the 1,9-dideoxy derivative of forskolin, or 8-bromocyclic AMP on cyclic AMP and melatonin production in the Syrian hamster pineal gland in organ culture

The possible involvement of cyclic AMP in the regulation of melatonin production was investigated in cultured Syrian hamster pineal glands. Addition of forskolin, an adenylate cyclase activator, to the incubation medium caused marked increases in both cyclic AMP and melatonin levels in glands collected in the second half of the dark period. 8-Bromocyclic AMP, an analogue of cyclic AMP, also increased melatonin production. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was ineffective in stimulating either cyclic AMP levels or melatonin production. These results support a primary role for cyclic AMP in the nocturnal increase of melatonin production in the Syrian hamster pineal gland.     

Histamine may act through cyclic AMP on hippocampal neurones

Bath applied histamine and 8-bromo-cyclic AMP and intracellularly injected cyclic AMP block-long-lasting afterhyperpolarizations and the accommodation of firing in CA1 pyramidal cells recorded in rat hippocampal slices. This action is due to reduction of a calcium-activated potassium conductance (gK(Ca)) and leads to potentiation of excitatory signals including epileptiform discharges. The effects are further potentiated and prolonged by a phosphodiesterase inhibitor (Ro 20-1724).     

GABA(B) receptor activation protects GABA(A) receptor from cyclic AMP-dependent down-regulation in rat cerebellar granule cells.

Interaction between GABAA and GABA(B) receptors was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. Our data show that the GABA(B) agonist (-)baclofen is not able, per se, to significantly change the muscimol-activated chloride current. However, (-)baclofen dose-dependently prevents the reduction of GABA(A) receptor function by forskolin, an activator of adenylate cyclase. The effect of baclofen is mediated by a pertussis toxin-sensitive G protein. In fact, in cells treated with pertussis toxin, baclofen and forskolin, the toxin is able to block baclofen action, allowing forskolin to act fully. The protective effect by GABA(B) receptor activation under these circumstances is most probably related to the prevention of cyclic AMP increases after forskolin treatment. In fact, in these neurons cyclic AMP and protein kinase A activation result in a down-regulation of GABA(A) receptor function. On the whole, the data indicate the presence of complex modulation of GABA(A) receptors by GABA(B) receptor types in cerebellum granule cells.     

G-protein mediating the slow depolarization induced by FMRFamide in the ganglion cells of Aplysia.

Application of FMRFamide (Phe-Met-Arg-Phe-NH2) induced a slow depolarization in neurons of the Aplysia abdominal ganglion. In voltage-clamped cells, FMRFamide induced a slow inward current that increased when the membrane was depolarized beyond -85 mV, showing a negative slope conductance. However, this inward current never reversed to outward current when hyperpolarized beyond the equilibrium potential for K+. The FMRFamide-induced response was markedly augmented in Ca(2+)-free media, but depressed in Na(+)-free media. It was unaffected by a change in external potassium. Intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) significantly depressed the FMRFamide response in a dose-dependent way. Injection of cholera toxin (CTX) which did not cause any current response, selectively and irreversibly blocked the FMRFamide response. Neither 3'-deoxyadenosine, an inhibitor of adenylate cyclase, nor H-8, an inhibitor of cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent kinase, depressed the FMRFamide response. 3-Isobutyl-1-methylxanthine (IBMX) did not augment the FMRFamide response appreciably. The FMRFamide response was not occluded at all by a relatively large injection of 8-bromo-cyclic AMP. It was concluded that the FMRFamide response is produced by the opening of the voltage-dependent Na(+)-channels via activation of a certain CTX-sensitive G-protein which is different from conventional "Gs" that activates adenylate cyclase.     

Cyclic AMP induces a voltage-dependent current in neurones of Aplysia californica

A voltage-dependent inward current is elicited by intracellular application of cyclic AMP in some neurons of Aplysia. The current has a slow time course and is present only at potentials more depolarized than -30 mV. Two other methods of increasing cellular cyclic AMP (phosphodiesterase inhibitors and sodium fluoride activation of adenylate cyclase) also induce an inward current at depolarized potentials.     

The Role of Intracellular cAMP in Mediating the Synchronizing Action of Noradrenaline on the Evoked Release of Quanta of Mediator in the Frog Synapse

Experiments on frog neuromuscular junction preparations with extracellular recording of action currents in nerve endings and single-quantum currents from endplates were used to assess the time course of evoked quantum mediator secretion by analyzing histograms showing the distribution of true synaptic delays. Studies using the cyclic AMP analog dibutyryl-cAMP (db-cAMP), the adenylate cyclase activator forskolin, and the nucleotide-dependent phosphodiesterase inhibitor isobutylmethylxanthine, showed that these agents, like noradrenaline, altered the kinetics of secretion of quanta, leading to synchronization of the release of mediator. After preliminary treatment of the neuromuscular preparation with db-cAMP, forskolin, or isobutylmethylxanthine, noradrenaline did not induce the synchronization of mediator release in quanta. It was concluded that the action of noradrenaline on the time course of secretion is mediated by activation of presynaptic receptors, increased adenylate cyclase activity, and increases in intracellular cAMP levels.     

Presynaptic facilitation of excitatory postsynaptic potential by glucagon in neurons of rat ventromedial hypothalamic slices.

Intracellular recordings were made from neurons in rat ventromedial hypothalamus (VMH), in vitro. Application of glucagon (100 nM to 5 microM) for 2-5 min increased the amplitude of excitatory postsynaptic potential (EPSP) lasting for 10-20 min. Forskolin and 8-bromo-cyclic AMP mimicked glucagon in producing a long-lasting facilitation of the EPSP. These drugs did not affect depolarizing response produced by glutamate. 3-Isobutyl-1-methylxanthine (IBMX) potentiated the time course of glucagon-induced facilitation of the EPSP. These results suggest that glucagon facilitates the EPSP probably by increasing transmitter release through activation of adenylate cyclase.     

Afterpotentials following penicillin-induced paroxysmal depolarizations in rat hippocampal CA1 pyramidal cells in vitro

Epileptic discharges were induced by superfusion of rat hippocampal slices with penicillin. Under these conditions the neurons generated paroxysmal depolarization shifts (PDS) after electrical stimulation of Schaffer collaterals. The PDS were followed by large afterhyperpolarizations lasting about 2 s. The mechanisms causing these afterhyperpolarizations were studied in CA1 pyramidal cells. A late component of the after hyperpolarizations, which determined their overall duration, was blocked by intracellular application of EGTA and reduced by superfusion with 8-Br-cAMP. In the same neurons these drugs had a comparable effect on after hyperpolarizations following depolarizing current injections; it was therefore concluded that the late component of the PDS afterhyperpolarizations was caused by a slow Ca²⁺-activated K⁺ current. An initial fast component of PDS afterhyperpolarizations, which peaked about 60 ms after PDS onset, was reduced by EGTA but not affected by 8-Br-cAMP suggesting that the fast Ca²⁺-activated K⁺ current also contributed to the PDS afterhyperpolarizations. Superfusion of the slice with the -aminobutyric acid B receptor (GABA_B) antagonists phaclofen or 5-aminovalerate reduced the amplitude of the afterhyperpolarizations during the first 1000 ms but did not affect the late Ca²⁺-dependent component, indicating that a GABA_B-mediated K⁺ inhibitory postsynaptic potential (IPSP) contributed to the PDS afterhyperpolarization. Intracellular injection of Cl^– revealed that an early part of the afterhyperpolarizations lasting about 500 ms was Cl^–-dependent. This component was blocked by superfusion of the slices with bicuculline, suggesting that a GABA_A-mediated Cl^– IPSP contributed to the PDS afterhyperpolarization. The experiments show that different synaptic and intrinsic components with different time courses participate in the generation of PDS afterpotentials.     

Involvement of adenylate cyclase inhibition in dopamine autoreceptor regulation of tyrosine hydroxylase in rat nucleus accumbens

In homogenates of rat nucleus accumbens, quinpirole, a dopamine (DA) D2 receptor agonist, inhibited the activation of tyrosine hydroxylase (TH) elicited by either forskolin, an activator of adenylate cyclase, or rolipram, a cyclic nucleotide phosphodiesterase inhibitor. The inhibition produced by 1 microM quinpirole was completely antagonized by the D2 blocker L-sulpiride (2 microM). Quinpirole failed to inhibit the stimulation of TH elicited by dibutyryl cyclic AMP (2 mM), which acts independently of adenylate cyclase. Quinpirole (10 microM) significantly inhibited the stimulation of adenylate cyclase activity elicited by 1 microM forskolin. These results indicate that mesolimbic DA autoreceptors can regulate TH activity by inhibiting a presynaptic adenylate cyclase system.     

Actions of acetylcholine and nicotine on rat locus coeruleus neurons in vitro

Intracellular recordings were made from neurons of the rat locus coeruleus in a slice of tissue cut from the pons and superfused in vitro at 37 degrees C. Both acetylcholine, in the presence of a muscarinic antagonist, and nicotine depolarized locus coeruleus neurons and increased the rate of action potential discharge. These effects persisted in a superfusing solution that contained no calcium ions and a raised magnesium ion concentration; because such solutions block synaptic potentials in the locus coeruleus, it was concluded that the acetylcholine and nicotine acted directly on the cell from which the recording was made. Acetylcholine-evoked depolarizations were blocked by hexamethonium or dihydro-beta-erythroidine, but not by alpha-bungarotoxin. Recordings of membrane current showed that acetylcholine and nicotine caused a net inward current, the amplitude of which increased linearly as the membrane potential was changed from -40 to -90 mV; the extrapolated reversal potential was about -20 mV (extracellular potassium concentration was 2.5 mmol/l). Depolarizations caused by nicotine declined during the presence of the agonist when the period of superfusion was continued for more than 2 min; repeated applications of nicotine evoked reproducible depolarizations only when the interval between them was at least 1 h. It is concluded that neurons of the rat locus coeruleus have nicotinic receptors with properties similar to those in peripheral ganglia with respect to reversal potential for the ion channel, insensitivity to alpha-bungarotoxin and propensity to desensitize.     

Forskolin prolongs action potential duration and blocks potassium current in embryonic chick sensory neurons

To determine if alterations in internal cyclic adenosine monophosphate (cAMP) play a role in modulation of voltage-dependent channels in embryonic chick sensory neurons in vitro, forskolin (a direct activator of adenylate cyclase) was tested on the cells. Forskolin, in concentrations between 1 and 100 M, produced dose-dependent, reversible increases in action potential duration. This effect of forskolin was blocked by incubation of the neurons in 1 mM 2,5-dideoxyadenosine, an inhibitor of forskolin-induced activation of cyclase in other cells. This suggests that the increase in action potential duration is likely to be mediated by activation of adenylate cyclase. Cholera toxin, another cyclase activator, also increased action potential duration when applied to the sensory neurons in a concentration of 10 g/ml. Forskolin applied to voltage-clamped neurons decreased a voltage-dependent outward current, a result consistent with its effect on the action potential. These effects of forskolin are mimicked by capsaicin, but are in marked contrast to those previously reported for norepinephrine on the action potential and membrane currents (Dunlap and Fischbach 1981). Furthermore, forskolin does not block (or attenuate) the effects of norepinephrine, suggesting that increases in adenylate cyclase activity are most likely not involved in norepinephrine's action on the calcium channel.     

Acetylcholine inhibits positive inotropic effect of cholera toxin in ventricular muscle

Acetylcholine (ACh, 10(-6) M) had no effect on basal adenylate cyclase activity (3.4 +/- 0.56 pmol cyclic AMP . min-1 . mg wet wt-1), adenosine 3',5'-cyclic monophosphate (cyclic AMP) content (0.88 +/- 0.09 pmol/mg wet wt), or the force of contraction in paced (2.5 Hz) chick embryo right ventricles superfused with Tyrode solution. After 60-180 min of superfusion in the presence of cholera toxin (5 x 10(-6) g/ml), adenylate cyclase activity (1.7 times), cyclic AMP content (2.4 times), and contractility (2.4 times) had increased significantly above basal levels. ACh reversed the positive inotropic effect of cholera toxin but did not change the increased activity of adenylate cyclase and content of cyclic AMP obtained in cholera toxin. Stimulation of adenylate cyclase by isoproterenol (ISO) was inhibited by ACh in the absence and presence of cholera toxin. ACh did not change guanosine 3',5'-cyclic monophosphate (cyclic GMP) content in the absence or presence of cholera toxin. Cholera toxin has actions on chick embryo ventricle similar to those of the beta-adrenergic agonist, ISO, and the phosphodiesterase inhibitor, isobutylmethylxanthine. The ability of ACh to reverse the positive inotropic effect of cholera toxin without preventing the accumulation of cyclic AMP may involve the prevention or reversal of cyclic AMP-dependent phosphorylation. In this regard, reduction of Ca2+ influx through voltage-sensitive membrane channels may be an essential component of muscarinic inhibition.     

The D1 dopamine receptor agonist SKF-38393 stimulates the release of glutamate in the hippocampus

The present study was undertaken to better assess the role of dopamine on exocytosis. Since direct activation of adenylate cyclase (e.g., with forskolin) enhances neurotransmitter release it was of interest to see whether the activation of D1-type dopamine receptors, which are positively coupled to adenylate cyclase, could also modulate the molecular machinery underlying the fusion of synaptic vesicles and the release of neurotransmitter. To answer this question we have looked at the effect of the D1-type dopamine receptor agonist SKF-38393 on the spontaneous release of glutamate from cultured rat hippocampal neurons. SKF-38393 enhanced the frequency but not the amplitude of tetrodotoxin-resistant excitatory postsynaptic currents which argues for a presynaptic locus of D1 action. This effect was blocked by the D1-dopaminergic receptor antagonist SCH-23390 and the protein kinase A inhibitors H-7 and Rp-cAMP whereas pertussis toxin failed to affect the dopaminergic response. In addition, carbachol and Ruthenium Red also stimulated exocytosis but did not occlude the SKF-38393-induced modulation. These results indicate that SKF-38393 presynaptically enhances the release of glutamate via a pertussis toxin-insensitive and protein kinase A-dependent mechanism, which most likely involves D1-type dopamine receptors. Our results underline the importance of protein kinase A as potent modulator of synaptic transmission and suggest that high concentrations of dopamine can greatly enhance the release of glutamate in the hippocampus.     

Prostaglandin E(2) enhances axonal transport and neuritogenesis in cultured mouse dorsal root ganglion neurons

The effects of prostaglandin E₂ on axonal transport in cultured mouse dorsal root ganglion neurons were investigated by analysing the number of axonally transported particles under video-enhanced microscopy. Application of prostaglandin E₂ increased the number of particles transported in anterograde and retrograde directions. The EP₂ prostaglandin receptor agonist butaprost mimicked the effect of prostaglandin E₂, but the EP₁/EP₃ prostaglandin receptor agonist 17-phenyl trinor prostaglandin E₂ and the EP₃ prostaglandin receptor agonist M&B 28767 had no effect. The membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP and the adenylate cyclase activator forskolin mimicked the effect of prostaglandin E₂. The protein kinase A inhibitor H-89 reversibly reduced the number of particles in both anterograde and retrograde directions. The effects of prostaglandin E₂ and dibutyryl cyclic AMP were blocked by H-89. Taken together with previous biochemical studies showing that prostaglandin E₂ increases cyclic AMP levels, the present results suggest that prostaglandin E₂ enhances axonal transport via the EP₂ receptor and cyclic AMP-dependent protein kinase A pathway. We further investigated the role of prostaglandin E₂ in neurite growth. Prostaglandin E₂ increased both the number of cells exhibiting neurites and the neurite growth rate, operating by a similar mechanism to stimulation of axonal transport.

Prostaglandin E₂ may modulate axonal transport to supply materials for morphogenesis as well as other functions in sensory neurons.     

Phorbol ester-induced inhibition of the beta-adrenergic system in pulmonary endothelium: role of a pertussis toxin-sensitive protein

To investigate possible cellular mechanisms for how activation of protein kinase C inhibits the relaxation caused by isoproterenol, we studied the effect of the protein kinase C activator 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA) on the increase in cyclic AMP (cAMP) production and adenylate cyclase activity caused by isoproterenol in bovine pulmonary artery endothelial cells. Treatment of intact cells with PMA prevented in a time- and dose-dependent manner the increase in cAMP production caused by isoproterenol, whereas 4 alpha-phorbol-12 beta-myristate-13 alpha-acetate (4 alpha-PMA), which does not activate protein kinase C, did not affect isoproterenol-induced cAMP production. PMA also reduced the increase in adenylate cyclase activity caused by isoproterenol, forskolin, and Gpp(NH)p. To test the hypothesis that the inhibitory effect of PMA is mediated via a pertussis toxin-sensitive G protein, we determined whether pretreatment of the cells with pertussis toxin would prevent the inhibitory effects of PMA. In pulmonary endothelial cells, pertussis toxin ADP-ribosylated an Mr 40,000 peptide that comigrated with the pertussis toxin substrate of human erythrocytes. Pertussis toxin treatment eliminated the inhibitory effect of PMA on isoproterenol-stimulated cAMP production and adenylate cyclase activity. Thus, the protein kinase C activator PMA inhibits the increase in cAMP production and adenylate cyclase caused by isoproterenol. This inhibitory effect in endothelial cells appears to be mediated via a pertussis toxin-sensitive protein.     

Interleukin 1-induced lymphocyte binding to endothelial cells. Role of cAMP as a second messenger

Interleukin 1 (IL 1) is a potent protein mediator of inflammation. Among other things it increases the number of lymphocytes adhering to endothelial cell monolayers. We analyzed the signal transduction during IL 1-induced lymphocyte binding. Dibutyryl cyclic AMP, which is a cAMP analog able to penetrate into the cytosol, increased lymphocyte binding to the same extent as IL 1. Direct activation of adenylate cyclase by forskolin enhanced also lymphocyte binding. IL 1 increased the level of cytosolic cAMP in a time- and dose-dependent manner measured with radioimmunoassay. 2',5'-Dideoxyadenosine, which is an inhibitor of adenylate cyclase, decreased both the IL 1-induced lymphocyte binding to endothelial cells and elevation in cytosolic cAMP levels. Lymphocyte binding increased with cytosolic cAMP levels in accordance with elevation of IL 1 concentration. These results suggest that cAMP is essential in signal transduction during IL 1-induced lymphocyte binding to cultured endothelial cell monolayers.