Supersensitivity in rat caudate nucleus: Effects of 6-hydroxydopamine on the time course of dopamine receptor and cyclic AMP changes

The intranigral administration of 6-hydroxydopamine resulted in the destruction of dopaminergic nerve terminals in the rat caudate nucleus and a 98% decrease in dopamine content. The time courses of the effects of this treatment on dopamine stimulated cyclic 3′,5′-AMP accumulation in slices of caudate nucleus and on dopamine receptors in two behaviorally distinct denervation syndromes were determined in an investigation of the mechanism underlying supersensitivity in this system. The density of dopamine receptors was determined by measuring the high affinity binding of the dopamine receptor antagonist [³H]haloperidol. The density of dopamine receptors was decreased 4 days after the lesion surgery and this effect was probably due to the loss of presynaptic receptors. The density of dopamine receptors and the accumulation of cyclic AMP then increased, with a slower time course, reaching peak levels 10 days after lesioning. The maximal increase in density of dopamine receptors was 70% in both denervation syndromes, while the maximal increase in dopamine-stimulated cyclic 3′,5′-AMP levels was 300% at maximally stimulating concentration. The equilibrium dissociation constant (K_d) for haloperidol remained unchanged for 3 weeks following denervation, but there was a slight increase inK_d 40 days post-surgery. The turning behaviour in both syndromes was correlated with a decrease in dopamine levels. The present results are consistent with the notion that the supersensitivity to dopamine that occurs in caudate nucleus following 6-hydroxydopamine lesions has both pre- and post-synaptic components in both syndromes.     

Dopamine blocks carrier-mediated release of GABA from retinal horizontal cells

Incubation of isolated goldfish retinas with 5 mM glutamate or aspartate causes release of [³H]-GABA from H1 horizontal cell somata and dendrites (HSD) as determined by light microscopic autoradiography. This release of [³H]GABA occurs via a sodium-dependent transport carrier and can be blocked by dopamine. The effect of dopamine is mimicked by dibutyryl-cyclic AMP, indicating that it is due to an increase in the intracellular level of cyclic AMP in H1 cells. We suggest that the transmitter release of GABA from H1 cells occurs via a transport carrier which is regulated by an EPSP-like excitatory input from red cones and by an unconventional type of inhibition from dopaminergic interplexiform cells.     

Fencamfamine modulates sodium, potassium-ATPase through cyclic AMP and cyclic AMP-dependent protein kinase in rat striatum

Summary. Dopamine (DA) and fencamfamine (FCF) modulatory action on Na,K-ATPase and Mg-ATPase activity were evaluated in rat striatum. DA and FCF induced a decrease in Na,K-ATPase, without affecting Mg-ATPase activity. The effect of FCF was dose-dependent from 10 to 100 μM, with an IC₅₀ of 4.7 × 10⁻⁵ M. Furthermore, the effect of FCF (100 μM) increasing AMPc levels, but not GMPc, was nonadditive with that of DA (10 μM), which is consistent to a common site of action. The 8-bromo-cyclic AMP also induced a specific reduction in the Na,K-ATPase activity. The reduction of Na,K-ATPase induced by FCF (100 μM) was blocked by either SCH 23390 or sulpiride, which are D1 and D2 receptor antagonists. The decrease in striatal NA,K-ATPase activity induced by FCF was blocked by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), but not by KT 5823, a selective inhibitor of cyclic GMP-dependent protein kinase (PKG). Otherwise, KT 5720 or KT 5823 did not produce any change in Na,K-ATPase or Mg-ATPase activity. These data suggest that FCF reduces Na,K-ATPase activity through cyclic AMP-dependent changes in protein phosphorylation via a PKA mechanism. Accepted January 20, 1998; received June 2, 1997     

Effects of intraocular injections of 6-hydroxydopamine on dopamine-dependent cyclic AMP accumulation in intact pieces of carp retina

Dopamine-stimulated cyclic AMP accumulation was measured in intact pieces of carp retina following intraocular injections of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). This treatment is known to induce a selective destruction of dopaminergic nerve terminals, and in these experiments caused a 94% reduction in retinal dopamine content. However, dopamine-dependent cyclic AMP accumulation was essentially unaltered in retinas exposed to 6-OHDA, indicating that the dopamine receptors linked to adenylate cyclase in the carp retina are located mainly on postsynaptic elements, and not presynaptically on the dopaminergic terminals.     

Forskolin enhancement of acetylcholine-evoked cyclic AMP formation and catecholamine release in perfused dog adrenals

Summary Unstimulated efflux of cyclic AMP from perfused dog adrenal glands was not altered by 0.1M of forskolin and was slightly increased by 0.3 and 1.0M of forskolin. ACh stimulated efflux of cyclic AMP which preceded CA release and the efflux was dose-dependently enhanced by forskolin. Forskolin did not affect the spontaneous CA release but enhanced ACh-evoked catecholamine (CA) release. There was a close correlation between the dose relationship of forskolin enhancement of stimulated-cyclic AMP efflux and that of evoked-CA release. ACh-evoked CA release in the presence of forskolin was further potentiated by R020-1724, a phosphodiesterase inhibitor. CA release evoked by excess K⁺, or by caffeine in the presence or absence of external Ca²⁺ was also potentiated by forskolin. These results suggests that cyclic AMP generation may increase in response to stimulation of adrenal chromaffin cells and that the resulting increase of the nucleotide may function as a facilitating modulator of CA release.     

Antagonism of VIP-stimulated cyclic AMP formation in chick brain

Of eight peptides tested (0.01–5 μM), only two, that is, pituitary adenylate cyclase-activating polypeptide (PACAP27) and chicken vasoactive intestinal peptide (cVIP), potently stimulated cyclic AMP (cAMP) production in cerebral cortical slices of the chick. Mammalian VIP (mVIP) showed some activity only at the highest dose tested, whereas truncated forms of PACAP or VIP, that is, PACAP6-27, cVIP6-28, and mVIP6-28, or hybrid compounds, that is, neurotensin6-11-cVIP7-28 (NT-cVIP) and neurotensin6-11-mVIP7-28 (NT-mVIP), were inactive. Thirty-minute preincubation of chick cortical slices with 5 μM PACAP6-27, NT-cVIP, or NT-mVIP competitively antagonized the cAMP effects of cVIP (0.03–1μM), with the truncated form of PACAP being the best antagonist. Preincubation of slices with 5 μM mVIP6-28 also produced a significant inhibition of the cVIP (0.1–1 μM)-induced increase in cAMP production; however its action was independent of the concentration of cVIP. In contrast to mVIP6-28, cVIP6-28 showed no antagonistic activity against the full-length peptide. In parallel experiments, 30-min pretreatment of cortical slices with 5 μM PACAP6-27 significantly antagonized the PACAP38-evoked increase in cAMP formation, whereas mVIP6-28 or the NT-mVIP hybrid was ineffective. It has been concluded that in the chick brain, PACAP and cVIP stimulate cAMP biosynthesis via PAC₁ and VPAC-type receptors, respectively, and PACAP6-27 seems to be the most potent, yet PACAP/VIP receptor-nonselective antagonist. Unlike truncated PACAP, the NT-VIP hybrid peptides tested may represent VPAC-type receptor-selective blocking activity.     

Selective increase of R-I subunit of cyclic AMP-dependent protein kinase in glia-rich primary cultures upon treatment with dibutyryl cyclic AMP

Levels of cyclic GMP-dependent protein kinase and of the subunits (R-I, R-II and C) of cyclic AMP-dependent protein kinase were determined in two types of neural primary cell cultures that were either treated or not treated with dibutyryl cyclic AMP. Astroglia-rich cell cultures from newborn rat brain responded to exposure to dibutyryl cyclic AMP by a 2-3-fold increase in the level of R-I subunit, as demonstrated by two radioimmunological procedures, while the levels of the other subunits (R-II and C) and of cyclic GMP-dependent protein kinase remained unaffected. In contrast, neuron-rich cell cultures from embryonic rat brain did not display such a change in the level of R-I subunit. Thus, the elevation in the level of R-I elicited by dibutyryl cyclic AMP in normal non-malignant neural cells in culture was restricted to glial rather than neuronal cells.     

Adenosine-mediated regulation of cyclic AMP levels in isolated incubated retinas

The effect of adenosine on the modulation of retinal cAMP levels was assessed in several mammalian species including mouse, rat, guinea pig and rabbit. Adenosine had no effect when added to incubated rat, mouse and guinea pig retinas. However, levels of cAMP were elevated in dose-dependent manner by adenosine in both light- and dark-adapted incubated rabbit retinas. Isobutylmethylxanthine (IBMX) blocked the elevation elicited by adenosine. Norepinephrine and dopamine also elevated cAMP in incubated rabbit retinas and these effects were not blocked by IBMX. The elevations of cyclic AMP levels produced by adenosine were additive with the effects of dopamine or norepinephrine. These results indicate that an adenosine-sensitive cAMP system exists in rabbit retina, and it is probably distinct from the dopamine and norepinephrine regulated cyclic AMP systems.     

Alpha-1 adrenergic receptor binding and adrenergic-stimulated cyclic AMP production in rat cerebral cortex after chronic lithium treatment

Summary Administration to rats of dietary lithium for 30 days was followed by evaluation of alpha-1 adrenergic receptor binding and of adrenergic-stimulated cyclic AMP (cAMP) accumulation in rat cerebral cortex. Chronic lithium treatment did not alter the binding characteristics of [³H]prazosin or the proportion of alpha-1 adrenergic receptor subtypes distinguished by chlorethylclonidine (CEC) pretreatment in rat cerebral cortical membranes. Accumulation of cAMP in cortical slices incubated with adrenergic agonists was unaffected in lithium-treated rats. These results demonstrate that chronic lithium treatment-induced reduction of norepinephrine (NE)-stimulated phosphoinositide (PI) hydrolysis (Casebolt and Jope, 1987) is not due to changes in the alpha-1 adrenergic receptor and is more sensitive to in vivo lithium treatment than is adrenergic-stimulated cAMP accumulation.     

Effects of dopamine on cyclic AMP concentration in the anterior pituitary glandin vitro

Summary Effects of different concentrations of dopamine on the cyclic AMP concentration in the rat anterior pituitary gland were investigated in vitro. Low concentrations of dopamine (10^–9–10^–8 mol/l) were found to decrease, whereas the high concentration (10^–5 mol/l) increased the cyclic AMP concentration in pituitaries collected from ovariectomized and estradiol-treated females. In contrast, dopamine had no effect on the anterior pituitary cAMP concentration when pituitaries were collected from ovariectomized rats which had not received estrogen replacement. These data show that the action of dopamine on the anterior pituitary cAMP largely depends on the dopamine concentration and the hormonal state of animals.This paper was supported by the Polish Academy of Sciences Grant No. 10.4.2.01.5.5.     

Free cyclic AMP increases in PC12 cells on depolarization

The temporal dynamics of the intracellular second messenger cyclic AMP (cAMP) were monitored in living PC12 cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator. When the cells were depolarized by exposure to high K⁺, the free cAMP concentration was elevated, and then slowly decreased back to resting levels when the depolarizing stimulus was removed. Furthermore, the cAMP elevation due to depolarization decreased with successive depolarizations. © 1997 Wiley-Liss Inc.     

Increasing membrane polyunsaturated fatty-acid content augments cyclic AMP formation and prostaglandin production in NIE-115 neuroblastoma

1. Addition of linoleic acid (50 μM) to culture medium significantly increases levels of polyunsaturated fatty acids (PUFA) in membrane phospholipids of NIE-115 neuroblastoma.

2. Basal levels of cyclic AMP are elevated significantly in supplemented ceils.

3. Exogenous prostaglandins (PG) PGE₁ and PGD₂ stimulate cAMP formation in NIE-115 neuroblastoma.

4. Supplemented cells produce higher levels of PGE and PGD than do control cultures.

5. Inclusion of cyclooxygenase inhibitors in culture medium does not block elevation of cyclic nucleotide in supplemented cells.

6. Endogenous PG production and receptor activation cannot account for increased cAMP in EFA-supplemented neuroblastoma.     

Antagonism of VIP-stimulated cyclic AMP formation in chick brain

Of eight peptides tested (0.01-5 microM), only two, that is, pituitary adenylate cyclase-activating polypeptide (PACAP27) and chicken vasoactive intestinal peptide (cVIP), potently stimulated cyclic AMP (cAMP) production in cerebral cortical slices of the chick. Mammalian VIP (mVIP) showed some activity only at the highest dose tested, whereas truncated forms of PACAP or VIP, that is, PACAP6-27, cVIP6-28, and mVIP6-28, or hybrid compounds, that is, neurotensin6-11-cVIP7-28 (NT-cVIP) and neurotensin6-11-mVIP7-28 (NT-mVIP), were inactive. Thirty-minute preincubation of chick cortical slices with 5 microM PACAP6-27, NT-cVIP, or NT-mVIP competitively antagonized the cAMP effects of cVIP (0.03-1 microM), with the truncated form of PACAP being the best antagonist. Preincubation of slices with 5 microM mVIP6-28 also produced a significant inhibition of the cVIP (0.1-1 microM)-induced increase in cAMP production; however its action was independent of the concentration of cVIP. In contrast to mVIP6-28, cVIP6-28 showed no antagonistic activity against the full-length peptide. In parallel experiments, 30-min pretreatment of cortical slices with 5 microM PACAP6-27 significantly antagonized the PACAP38-evoked increase in cAMP formation, whereas mVIP6-28 or the NT-mVIP hybrid was ineffective. It has been concluded that in the chick brain, PACAP and cVIP stimulate cAMP biosynthesis via PAC1 and VPAC-type receptors, respectively, and PACAP6-27 seems to be the most potent, yet PACAP/VIP receptor-nonselective antagonist. Unlike truncated PACAP, the NT-VIP hybrid peptides tested may represent VPACtype receptor-selective blocking activity.     

Relationship between cyclic AMP and thromboxane formation in platelet-endothelial cell interactions

Human platelet aggregation was triggered in the presence of various numbers of cultured endothelial cells. Thromboxane B2 formation and platelet cyclic AMP were also measured. Using a low concentration of thrombin (0.025U/ml) as aggregating agent, the inhibition of platelet aggregation correlated with that of thromboxane formation and was directly related to both the number of endothelial cells and platelet cyclic AMP. In contrast, using arachidonic acid (10(-5)M) instead of thrombin, platelet aggregation could be abolished although thromboxane formation was not affected. These results suggest that platelet aggregation induced by low concentrations of thrombin might be dependent on prostaglandin endoperoxides/thromboxane A2 production which could be inhibited by cyclic AMP. The normal synthesis of thromboxane B2 from exogenous arachidonate indicates that cyclic AMP is only active upon the liberation of endogenous arachidonate from platelet phospholipids.     

Effects of neurotransmitters and cyclic AMP on somatostatin release from cultured cerebral cortical cells

The influence of cortical neurotransmitters and cyclic AMP on the release of immunoreactive somatostatin (IRS)from cultured cortical cells was examined. Cells were obtained by mechanoenzymatic dispersal of telencephalons of 17-day-old rat embryos and were maintained as monolayers in minimum essential medium with 10% heat-inactivated horse serum. After the cultures had stabilized morphologically and in cellular IRS content they were subjected to rapid sequential changes of a buffered salt solution with or without test substances added. The amount of somatostatin released was measured by a specific radioimmunoassay. Acetylcholine and the GABA antagonist, picrotoxin, both stimulated IRS release. The cholinergic stimulation was predominantly muscarinic. GABA and histamine, to a lesser extent, were inhibitory and norepinephrine and serotonin produced no net change in IRS release. Both cAMP and theophylline (DMX) stimulated IRS release. These results confirm the potential of intrinsic cortical somatostatinergic neurons to respond to endogenous neurotransmitters and further establishes somatostatin as a cortical neuromodulator.     

Spatiotemporal properties of cytoplasmic cyclic AMP gradients can alter the turning behaviour of neuronal growth cones

Growth cones, the terminal structures of elongating neurites, use extracellular guidance information in order to navigate to appropriate target cells. The directional information of guidance cues is transduced to a cytoplasmic gradient of messenger molecules across the growth cone leading to rearrangements of the cytoskeleton. One messenger molecule regulating growth cone turning is cAMP, which is also known to be sufficient to direct growth cone attraction. Cytoplasmic cAMP gradients have been generated in the present study by photolysing caged cAMP with UV light focused on one side of growth cones of chick sensory neurons. Using this method we show that only specific time patterns of pulsed cAMP release are capable of inducing growth cone turning whereas others, which release the same amount of cAMP, are ineffective. Theoretical calculations show that diverse time patterns produce different intracellular gradients, which were visualized directly in HeLa cells expressing cAMP-sensitive ion channels as a reporter system. Together these data indicate that the spatiotemporal properties of the intracellular gradient are crucial for growth cone turning.