Visualization of detergent insoluble cyclic AMP-dependent protein kinase RIalpha aggregates in the rat brain

Regulatory subunits of the cAMP dependent protein kinases are the most abundant receptor for cAMP in eukaryotic cells. Four isoforms of regulatory subunits (RIalpha and -beta, RIIalpha and -beta) have been distinguished. Distribution of the most abundant RII isoforms has been extensively studied in the brain, by immunohistochemistry and biochemical fractionation, while the least abundant RI isoforms have been neglected. In neurons most regulatory subunits are bound to the cytoskeleton. A protocol is presented that allows immunohistochemical and biochemical characterization of detergent-insoluble RI isoforms in the brain.     

Abnormalities of cyclic adenosine monophosphate signaling in platelets from untreated patients with bipolar disorder

BACKGROUND: Abnormalities in the cyclic adenosine monophosphate (cAMP)-dependent phosphorylation system have been recently reported in patients with bipolar disorder. We evaluated the immunoreactivity of the regulatory and catalytic subunits of cAMP-dependent protein kinase (protein kinase A) and 1 of its substrates, Rap1, in platelets from untreated euthymic, manic, and depressed patients with bipolar disorder and healthy subjects. METHODS: Platelets were collected from 112 drug-free patients with bipolar disorder (52 euthymic, 29 depressed, and 31 manic) and 62 healthy subjects. The levels of cAMP-dependent protein kinase and Rap1 were assessed by Western blot analysis, immunostaining, and computer-assisted imaging. RESULTS: The immunolabeling of the catalytic subunit of cAMP-dependent protein kinase was significantly different among groups (P<.001), with higher values in untreated depressed and manic patients with bipolar disorder compared with untreated euthymic patients with bipolar disorder and healthy subjects. No significant differences were found in the immunolabeling of the regulatory subunits (type I and type II) of cAMP-dependent protein kinase. The immunolabeling of Rap1 was significantly higher (P<.001) in untreated euthymic, depressed, and manic patients than in healthy persons. CONCLUSIONS: Levels of Rap1 and the catalytic subunit of cAMP-dependent protein kinase are altered in the platelets of bipolar patients. These findings may provide clues toward understanding the involvement of cAMP signaling in the pathogenesis of bipolar disorder.     

Excitation of locus coeruleus neurons by vasoactive intestinal peptide: role of a cAMP and protein kinase A

In accord with previous studies, it was found that vasoactive intestinal peptide (VIP), a powerful activator of adenylate cyclase, and cAMP-active agents (i.e., 8-Br-cAMP, forskolin, and Ro20-1724) increased the firing rate of noradrenergic neurons in the locus coeruleus (LC) by inducing an inward current. The response to VIP was usually more rapid and larger in a subpopulation of LC neurons with subthreshold rhythmic oscillations in membrane potential (oscillatory cells) as compared to nonoscillatory cells. In either case, the inward currents elicited by VIP and cAMP-active agents were found to be nonadditive, suggesting the action of VIP, at least in part, is via the same mechanism as that of cAMP-active agents. Intracellular application of a specific protein (or related peptide) inhibitor of cAMP-dependent protein kinase markedly attenuated the activation induced by either cAMP-active agents or VIP, suggesting that cAMP-dependent protein kinase (protein kinase A), presumably through protein phosphorylation, plays a role in the action of VIP. Taken together, the results provide evidence that cAMP and protein kinase A are involved in mediating the electrophysiological actions of VIP on LC neurons.     

(3)H]cAMP binding sites and protein kinase a activity in the prefrontal cortex of suicide victims.

OBJECTIVE: The cAMP-dependent enzyme protein kinase A phosphorylates intracellular proteins upon activation and thereby plays a major role in mediating various physiological functions in the brain. To examine the role of this enzyme in suicidal behavior, the authors examined the catalytic and regulatory activities of protein kinase A in the postmortem brain of suicide victims. METHOD: Brain tissues were collected from 17 suicide victims and 17 nonpsychiatric comparison subjects. Regulatory activity was determined by examining [(3)H]cAMP binding to protein kinase A, while catalytic activity was determined by enzymatic assay in the presence (total activity) and the absence (endogenous activity) of cAMP in the membrane and cytosol fractions of the prefrontal cortex. RESULTS: The number (B(max)) of [(3)H]cAMP binding sites to protein kinase A was significantly lower in the suicide victims without any changes in affinity in either the membrane or cytosol fractions of the prefrontal cortex. Further, significantly less protein kinase A activity, both in the presence and the absence of cAMP, was seen in the membrane and cytosol fractions of the prefrontal cortex of suicide victims; however, the difference in total protein kinase A activity was much more pronounced. CONCLUSIONS: The results suggest that cAMP binding to the regulatory subunits of protein kinase A, as well as the phosphotransfer catalytic activity of protein kinase A, are lower in the prefrontal cortex of suicide victims than in nonpsychiatric comparison subjects, which may be of clinical relevance in the pathophysiology of suicidal behavior.     

Effect of a catalytic subunit of cAMP-dependent protein kinase on acetylcholine-induced chloride currents in mollusk neurons]

It is shown that the amplitude of ACh-induced chloride currents decreases with introduction of cAMP in dialyzed neurons of Helix pomatia, the rate of desensitization of the acetylcholine receptors (AChR) being insignificantly changed. Introduction of an active catalytic subunit (c.s.) of cAMP-dependent protein kinase (cAMP-PK) mimics this effect. It is supposed that the influence of cAMP on the functional properties of the AChR is mediated by the activation of cAMP-PK and further phosphorylation of the AChR by the catalytic subunits of this protein kinase.     

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.     

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.     

Fetal development and neuronal/glial cell specificity of cAMP-dependent protein kinase subunit mRNAs in rat brain

All known actions of cAMP in the brain require cAMP-dependent protein kinase (cAMPdPK), which consists of regulatory (R) and catalytic (C) subunits (R2C2). Using homologous rat cDNAs for all known cAMPdPK subunit isoforms found in the brain (RI alpha, RI beta, RII alpha, RII beta, C alpha, C beta) we observe that, in the fetal rat brain from 12 days of gestation to birth, while alpha subunit (RI alpha, RII alpha, C alpha) mRNA levels are abundant, beta subunit (RI beta, RII beta, C beta) mRNA levels increase from undetectable or very low levels to abundant levels. Furthermore, while alpha subunit mRNA levels are abundant in both primary neuronal and primary glial cultures, beta subunit mRNA levels are very low (C beta) or undetectable (RI beta, RII beta) in primary glial cultures, but are abundant in primary neuronal cultures. Thus, prior to about 12 days of gestation, cAMP in the brain may act only via the alpha cAMPdPK subunits in neuronal and glial precursor cells. After 12 days of gestation, coincident with the onset of final cell division in neurons, beta cAMPdPK subunits may also mediate the effects of cAMP predominantly in neurons.     

Aggregates of cAMP-dependent kinase RIalpha characterize a type of cholinergic neurons in the rat brain

Acetylcholine is synthesized by different types of neurons, showing a distinct biochemical phenotype. Aggregates of RIalpha regulatory subunit of cAMP-dependent protein kinases are visualized by immunohistochemistry only in some cholinergic neurons, since they tightly colocalize with two different markers, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). These neurons are present mainly in brain areas related to the limbic system. None of the other regulatory subunits of cAMP dependent kinases colocalize with cholinergic markers.     

Compartmentalization of adenosine 3′:5′-monophosphate-binding proteins in human platelets

Adenosine 3′:5′-monophosphate (cAMP) plays a major role in the control of platelet function. The major cAMP-binding proteins in mammalian tissues, the regulatory subunits of the cAMP-dependent protein kinases, were evaluated in human platelets with the photoaffinity label 8-azido [³²P]cAMP. We observed that approximately equal quantities (0.408 – 0.421 pmol/mg protein) of the regulatory subunit of a type I cAMP-dependent protein kinase (R_I) were present in the particulate and soluble fractions from human platelets.The soluble fraction contained 0.351 pmol/mg protein of the regulatory subunit of a type II cAMP-dependent protein kinase (R_II). However, little if any, R_II was present in the particulate fraction. These data indicate that the type II cAMP-dependent protein kinase is compartmentalized to the soluble fraction and suggest that preferential phosphorylation of protein kinase substrates may result from such compartmentalization.The K_D for cAMP-binding to R_I was similar in the soluble and particulate fractions (0.005 μM and 0.003 μM, respectively). The K_D for cAMP-binding to R_II varied widely. In some experiments, a clear-cut, biphasic double-reciprocal plot was observed, suggesting two different cAMP-binding sites. Two-dimensional analysis of 8-azido-[³²P]cAMP-labeled platelets prepared in a buffer which inhibits platelet phosphoprotein phosphatase suggested the presence of equal amounts of two forms of R_II with characteristics similar to those previously described for phospho- and dephospho-R_II.     

Cyclic AMP, but not basic FGF, increases the in vitro survival of mesencephalic dopaminergic neurons and protects them from MPP(+)-induced degeneration.

We studied how stimulation of protein kinase C and cAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differentiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and cAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular cAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl cAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results suggest that increased intracellular levels of cAMP protect dopaminergic neurons in situations of stress like the process of dissociation and plating or the exposure to neurotoxic compounds. Our results reveal novel possibilities for the treatment of Parkinson's disease.     

Lidocaine suppresses the sodium current inEuhadra neurons which is mediated by cAMP-dependent protein phosphorylation

The action of a local anesthetic, lidocaine, in association with the cyclic AMP (cAMP)-mediated intracellular biochemical process, was examined in identifiedEuhadra neurons. Lidocaine dose-dependently inhibited the inward current which was elicited by dibutyryl cAMP (db-cAMP) and isobutylmethylxanthine (IBMX). This inhibitory effect was transiently reversed by the intracellular injection of a catalytic subunit of a cAMP-dependent protein kinase. The inward current elicited by db-cAMP and IBMX was abolished by Na⁺-free saline but not by Ca²⁺-free saline. The data suggest that lidocaine is not acting directly on the Na⁺ channel, but acts at a level proximal to the catalytic subunit of cAMP-dependent protein kinase.     

Facilitatory transmitter causes a selective and prolonged increase in adenosine 3':5'-monophosphate in sensory neurons mediating the gill and siphon withdrawal reflex in Aplysia

Sensitization of the gill and siphon withdrawal reflex in the marine mollusc, Aplysia california, is a simple form of learning Underlying this behavioral changes is a cascade of biochemical events. The first step in this cascade is postulated to be an increase in cAMP within the sensory neurons of the abdominal ganglion. We have developed a labeling protocol with 32Pi which permits us to measure the synthesis of cAMP within a single sensory neurons. Application of serotonin for 5 min was found to triple the content of [32P]cAMP in sensory neurons. The response is specific to serotonin: dopamine, a transmitter that does not produce sensitization, did not increase cAMP. Physiological stimulation of facilitator neurons also resulted in a 3.5-fold increase of cAMP in sensory neurons but not in other cells of the ganglion. We studied the time course of the increase of cAMP in sensory cells stimulated with serotonin and found that it parallels closely the time course of the short term form of presynaptic facilitation. We also have determined the effects of transmitters on the synthesis of cAMP in other identified neurons of the ganglion. The bag cells responded specifically to serotonin. R15, which has been shown to be hyperpolarized both the serotonin and by dopamine, responded to both transmitters by increased synthesis synthesis of cAMP. Thus, the dopamine- and serotonin-sensitive cyclase can be localized to both the same and different cells. Other cells did not respond to serotonin or to dopamine, indicating that a transmitter-sensitive adenylate cyclase is a specific property and is not present in all neurons.     

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.     

Calcium- and cyclic nucleotide-dependent protein kinases and their substrates in the Aplysia nervous system

Homogenates of the Aplysia nervous system contain protein kinase activities sensitive to cAMP, cGMP, and Ca2+/calmodulin. The cAMP- and cGMP-dependent activities are either soluble enzymes or are only loosely bound to membranes, since they can be detected only in crude but not in washed membrane fractions, and are present in 20,000 or 100,000 X g supernatants prepared from homogenates. In contrast there are both soluble and tightly membrane-bound Ca2+/calmodulin-dependent protein kinase activities. The three activities present in supernatant fractions can be separated by chromatography on DE-cellulose, indicating that they are due to distinct enzyme species. Substrates for these enzymes were analyzed by two-dimensional gel electrophoresis. Protein phosphorylation within the identified Aplysia neuron R15 in vivo was measured by the intracellular injection of [gamma-32P]ATP. cAMP stimulates the phosphorylation of nine proteins and decreases phosphorylation of two proteins in this cell. This in vivo pattern was compared with in vitro phosphorylation measured in homogenates of whole ganglion. Most of the phosphoproteins affected by cAMP in neuron R15 in vivo are also substrates for cAMP-dependent protein kinase in vitro. Thus, the in vitro system will be a useful tool for detailed biochemical analysis of phosphoproteins which have been identified as being physiologically relevant in vivo.     

Ontogenetic changes in the cyclic adenosine 3',5'-monophosphate-stimulatable phosphorylation of cat visual cortex proteins, particularly of microtubule-associated protein 2 (MAP 2): effects of normal and dark rearing and of the exposure to light

Based on a theory that a norepinephrine-stimulated cascade of events resulting in an increase of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) modulates the state of plasticity for the receptive field property of visual cortical neurons, we have followed the ontogenetic changes in cAMP-stimulated phosphorylation of proteins in whole homogenates obtained from developing visual cortices of cats. In vitro phosphorylation was assayed with and without cAMP and the cAMP-dependent protein kinase, and the phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were counted for 32P incorporated from [gamma-32P]ATP. It was found that the regulatory subunits of the cAMP-dependent protein kinase are present and fully active by birth, whereas the synapsin content increases at a rate concomitant with synaptogenesis. These ontogenetic developments are not influenced by dark rearing (DR) from birth, a procedure which postpones the onset of the critical period (CP) for plasticity. By contrast, the cAMP-stimulatable phosphorylation of microtubule-associated protein 2 (MAP 2), which under normal rearing conditions increases from birth to the second month, is strongly modulated by the presence of light in the environment. After DR for various periods, kittens were subsequently exposed to light so as to trigger the onset of the CP that had been postponed. A few hours of light were sufficient to cause a large increase in the in vitro phosphorylation of MAP 2. This effect is not observed in the auditory cortex or the lateral geniculate nucleus of the same animals, or in the visual cortex of normally reared cats which were then dark reared in adulthood. But this effect was seen in the visual cortices of cats following 5 months of DR from birth, animals which by chronological age have passed the CP, presumably because the onset of the CP was extended by the DR procedure. The cAMP-dependent phosphorylation of MAP 2 (and its dephosphorylation) may be an important factor for determining the state of plasticity in the CP through its affecting the dendritic cytoskeletal organization involving tubulin and actin.