Application Of db-cAMP Research Articles

Pharmacogenetic approaches in altering heart rate in Drosophila larvae

Fruit fly, Drosophila melanogaster, is a good model organism to study the underlying ionic mechanism of heart rate (HR) regulation. It is already known that many neuromodulators (serotonin, dopamine, octopamine, acetylcholine) change the HR in larvae. We used genetic and pharmacological approaches to investigate the extent to which cAMP‐PKA signaling mediates the effects of serotonin (5‐HT) and dopamine (DA) on larval HR. One approach involved the use of transgenic flies that express designer receptors exclusively activated by designer drugs (DREADDs). These engineered G‐protein coupled receptors helped to demonstrate that the effect of 5‐HT, which increases HR, could be overridden by the action of Gái which in turn inhibits adenylyl cyclase (AC). Moreover, activation of AC by forskolin increased HR. However, application of dbcAMP (cAMP analogue, PKA activator) and SQ, 22,536 and ddadenosine (AC inhibitors) did not have a substantial effect on the HR. DA activates parallel signaling cascades in cardiac cells with the end result of increased HR. SKF38393 (a vertebrate D1 agonist) and quinpirole (a vertebrate D2 agonist) increase HR to various degrees. SCH23390 and spiperone (D1 and D2 antagonists) each inhibit the stimulatory effect of DA. And SQ22536 inhibits the effect of DA. Together these results indicate that serotonin and dopamine regulate fruit fly HR through canonical GPCR pathways.

Neuropeptide Y inhibits axonal transport of particles in neurites of cultured adult mouse dorsal root ganglion cells.

Neuropeptide Y (NPY) plays a modulatory role in processing nociceptive information. The present study investigated the effects of NPY on axonal transport of particles in neurites of cultured adult dorsal root ganglion (DRG) cells using video-enhanced microscopy. Application of NPY decreased the number of particles transported in both the anterograde and retrograde directions. This effect was persistently observed during NPY application and was reversed after washout. The inhibitory effect of NPY was concentration dependent between 10(-9) M and 10(-6) M. The instantaneous velocity of individual particles moving in anterograde and retrograde directions was also reduced by NPY. Both the NPY Y1 receptor agonist [Leu31,Pro34]-NPY and NPY Y2 receptor agonist NPY(13-36) mimicked the effect of NPY on the number of transported particles. An immunocytochemical study using an antiserum against the NPY Y1 receptor protein revealed that the Y1 receptor was expressed in the majority (85.9 %) of cultured adult mouse DRG cells. Pre-treatment of cells with pertussis toxin, a GTP-binding protein (G protein) inhibitor, completely blocked the inhibitory effect of NPY. Each application of SQ-22536, an adenylate cyclase inhibitor, and H-89, a protein kinase A inhibitor, mimicked and occluded the effect of NPY. In contrast, dibutyryl cAMP (dbcAMP), a membrane permeable cAMP analogue, and forskolin, an activator of adenylate cyclase, produced a transient increase in axonal transport. The application of dbcAMP and forskolin in combination with NPY negated the effect of NPY alone. These results suggest that NPY, acting at Y1 and Y2 receptors, inhibits axonal transport of particles in sensory neurones. The effect seems to be mediated by a pertussis toxin-sensitive G protein, adenylate cyclase, and protein kinase A pathway. Therefore, NPY may be a modulatory factor for axonal transport in sensory neurones.

The effects of cAMP on the excitability and responses of defensive behavior command neurons in the common snail evoked by sensory stimuli.

Experiments on snails showed that extracellular application of dibutyryl-cAMP (db-cAMP) or intracellular application of cAMP for 30 min evoked increases in excitability and synaptic facilitation in responses to sensory stimulation of defensive behavior command neurons LP11 and RP11. Excitability increased 45-60 min after the start of addition of db-cAMP or cAMP and remained elevated until the end of the experiment (3-4 h). Synaptic facilitation started 50-60 min after the onset of extracellular application of db-cAMP and remained detectable in the responses of neurons to tactile stimulation of the head for 1 h and to application of dilute quinine solution for 2-4 h. Application of db-cAMP produced no changes in responses to tactile stimulation of the foot or mantle ridge. Intracellular injection of cAMP induced facilitation of neuron responses only to weak quinine solutions. The responses of neurons to tactile stimulation of the head, foot, and mantle ridge did not change after injections of cAMP. It is suggested that cAMP is involved in the mechanisms controlling the excitability of neurons LP11 and RP11. In addition, cAMP is selectively involved in the postsynaptic mechanism inducing the transient stage of long-term facilitation of synaptic "inputs," which mediates excitation evoked by chemical stimuli. This set of effects of cAMP is similar to effects arising during the development of nociceptive sensitization and in response to serotonin.

Involvement of phosphorylation of β-subunit in cAMP-dependent activation of L-type Ca 2+ channel in aortic smooth muscle-derived A7r5 cells

We investigated the effect of intracellular cAMP on the gating kinetics of L-type Ca 2+ channel in an A7r5 smooth muscle-derived cell line using the whole-cell patch-clamp technique. Application of dibutyryl cyclic AMP (db-cAMP) to the cell increased the magnitude of Ca 2+ currents through L-type Ca 2+ channels ( I Ca), and shifted the current-voltage relationship ( I– V curve) for I Ca to the left. The magnitudes of maximum I Ca were 14.1 ± 0.7 before and 16.0 ± 1.1 pA/pF after application of 1 mM db-cAMP ( P < 0.05). The values of the half-activation potential ( V 1/2) of I Ca, estimated from activation curves, were −7.0 ± 0.8 mV before and −10.8 ± 1.0 mV after application of db-cAMP ( P < 0.05). In cells pretreated with 10 μM Rp-cAMPS (a specific inhibitor of PKA), db-cAMP affected neither the I– V curve nor the activation curve for I Ca. In cells pretreated with the antisense oligonucleotide for the β-subunit of L-type Ca 2+ channel, db-cAMP failed to enhance I Ca or alter the activation curve. On the other hand, in the cells pretreated with the nonsense oligonucleotide, application of db-cAMP caused an increase in magnitude of I Ca and shifted the activation curve to the left. Western blot analysis revealed that the pretreatment of cells with antisense oligonucleotide but nonsense oligonucleotide reduced the expression of the β-subunit of the L-type Ca 2+ channel. We conclude that the cAMP-dependent phosphorylation of the β-subunit potentiates the voltage dependency of the activation kinetics of the L-type Ca 2+ channel in A7r5 cells.

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet beta-cells.

Intracellular cAMP-dependent modulation of L-type Ca2+ channel activation in cultured rat islet beta-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca2+ current (ICa) showed a fast activation followed by a slow inactivation, and was sensitive to Ca2+ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak ICa in a concentration-dependent manner. Values of the half-activation potentials (V1/2), taken from activation curves for ICa, were -16.7 +/- 1.8 and -21.9 +/- 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet beta-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca2+ channels.

Oxidized LDLs But Not Native LDLs Augment Ba 2+ Currents Through L-Type Ca 2+ Channels of the A7r5 Smooth Muscle-Derived Cell Line

The whole-cell patch-clamp method was used on A7r5 smooth muscle-derived cell line, and Ba 2+ currents through Ca 2+ channels were recorded. The A7r5 cells showed voltage-dependent, long-lasting Ba 2+ currents which were markedly inhibited by nifedipine (10 μM). The magnitude of the maximum Ba 2+ current (I ba(max)) was augmented by an application of dbcAMP (1 mM), but not affected by TPA (80 nM). Noradrenaline (NA) at 100 μM caused an increase in the I ba(max) by 19.7% in the presence of phentolamine (10 μM). This effect was cancelled by Rp-cAMPs (10 μM). In the presence of propranolol (10 μM), NA tended to reduce the I ba(max). Application of Ox-LDLs at 100 μg protein/ml caused an increase in the I ba(max) by 15.7%, whereas native LDLs did not change the I ba(max). Rp-cAMPs was ineffective to the Ox-LDL action on the I ba(max). In the presence of Ox-LDLs, NA augmented the I ba(max) by 21.4% in the presence of phentolamine. These results suggest that Ox-LDLs activate L-type Ca 2+ channels of A7r5 cells by a mechanism independent of cAMP/PKA signalling.

Cyclic AMP Does Not Induce the Down-Regulation of Calcium Current in Molluscan Neurons Through Kinase A Activation or Cytoplasmic Ca2+ Elevation

In isolated neurons of the land snail, a high threshold voltage-activated calcium current (ICa) was recorded using the two microelectrode voltage clamp method. Iontophoretic intracellular injection of cAMP or extracellular application of dbcAMP evoked a rapid and reversible decrease in peak amplitude of ICa in 30 out of 54 cells tested. The effects of cAMP on ICa and IBa were similar. cGMP injected into the same cell from another barrel of a multi-barreled micropipette did not mimic the effect of cAMP. The cyclic nucleotide phosphodiesterase activator, imidazole, caused an augmentation of ICa in 8 out of 13 cells tested. Intracellular injection of the Ca2+-chelator, EGTA, had no influence on the extent of cAMP-evoked depression of ICa. The inhibitors of kinase A, tolbutamide and H-8, evoked an effect resembling the effect of cAMP, i.e. depression of ICa. The results suggest that the down-regulation of ICa by cAMP is achieved through neither kinase A activation nor through cytoplasmic Ca2+ elevation.

Effects of ouabain, DBcAMP, caffeine, and high [Ca2+]o on twitch tension, intracellular Na+ activity, and action potential of guinea pig papillary muscles.

To explore ionic mechanisms of the positive inotropic process in guinea pig ventricular papillary muscles, we simultaneously measured twitch tension, intracellular Na+ activity (aiNa) and action potential at a stimulus rate of 60/min. The aiNa was 5.0 +/- 0.1 mM in the steady state. When the Na-K pump was inhibited by ouabain (1 microM), the twitch tension was increased in proportion to the aiNa, and the action potential was decreased at diastolic state and at duration for 90% of repolarization (APD90). While exposed to caffeine (3 mM), the aiNa kept increasing even when the twitch tension achieved a peak level. Concomitantly, the action potential slightly depolarized at diastolic state with a prolonged APD90. An application of DBcAMP (1 mM) or an increase in [Ca2+]o (from 1.8 to 3.6 mM) enhanced the twitch tension with a fall of aiNa and a shortening of APD90. These results suggest that in guinea pig papillary muscles Na-Ca exchange plays an important role in the regulation of contractile force and membrane potential, and that the Na influx should be balanced by the activation of the sarcolemmal Na-K pump, a negative feedback mechanism, to prevent calcium overload and abnormal pacemaker activity in electrical excitation.

β-Agonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the GΩ-seal whole cell clamp technique. In control, ACh (1 μM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 μM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolot (10 μM). Application of Db-cAMP (500 μM) mimicked the Iso effects. Intracellulary applied GTP-γS (100 μM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that β-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.

Research on the Differentiation of Human and Murine Neuroblastoma Cells

In vitro, we were able to induce a differentiation of human (SK-N-MC, IMR-32, Leo-2) and murine neuroblastoma cells (NA-2, C-1300, NIE-115) with dibutyryl cyclic 3'5'-adenosine monophosphate (dbcAMP), hypothalamic factor (HF), and somatostatin. As morphological criteria of cellular differentiation we used the decrease in cell proliferation and the formation of neurites. Functional parameters were the increase of A cholinesterase activity, cAMP level, and protein content, and the decrease of cGMP level. After application of dbcAMP and HF, the effects were stronger than after somatostatin. We believe that the action of HF and somatostatin is caused by an increase in cAMP levels. In the in vivo experiments, human and murine neuroblastoma cells (NA-2, C-1300, and Leo-2) were transplanted into nude/nude mice. After HF treatment of 14 mice with NA-2 tumors, 4 of the mice were tumor-free, and mean tumor weight was reduced to one-third of the controls. Of the animals with C-1300 and Leo-2 tumors, half became tumor-free, and mean tumor weight was reduced to one-fourth. The results indicate that the induction of cellular differentiation by factors and hormones may in future become a method of therapy for human neuroblastoma.

Calcium currents of neuroblastoma � glioma hybrid cells after cultivation with dibutyryl cyclic AMP and nickel

The long-term modulation of calcium (Ca2+) currents (ICa) was studied in 108CC15 neuroblastoma x glioma hybrid (NxG) cells grown under various culture conditions. The following results were obtained: 1. Addition of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP) or 0.1 microM forskolin to the culture medium increased a transient component of ICa two-fold within 3 days, from 21.0 +/- 1.6 pA/pF (n = 22) to a maximum of 40.0 +/- 2.6 pA/pF (n = 28). Under these conditions, cells also expressed a slowly inactivating ICa component (maximum after 3 days, 20.5 +/- 1.6 pA/pF, n = 28). 2. The fast inactivating ICa as well as the db-cAMP-induced slowly inactivating ICa were completely down-regulated during incubation of NxG cells with the inorganic Ca2+ channel blocker, nickel (Ni2+, 100 microM). The suppressing effect was reversed within 3 days of incubation in db-cAMP-containing medium lacking Ni2+. 3. Binding studies on membrane preparations of control and Ni2(+)-pretreated NxG cells revealed a marked difference in the maximal (+)3H-PN200-110 binding. The difference was seen in undifferentiated as well as in db-cAMP-incubated cells. 4. The protein synthesis blocker, cycloheximide, suppressed both the db-cAMP-induced increase and the reappearance of ICa following Ni2+ pretreatment. It is suggested that chronic application of db-cAMP or Ni2+ to NxG cells increases and decreases the number of Ca2+ channel proteins, respectively.

Redistribution of myosin during morphological reversion of Chinese hamster ovary cells induced by db-cAMP

Chinese hamster ovary (CHO) cells, an established, transformed line, regain several morphological and growth properties characteristic of normal fibroblasts when treated with dibutyryl cyclic AMP (db-cAMP). Untreated cells are compact, poorly spread, and covered with dynamic surface blebs. db-cAMP reversibly induces the elongation and flattening of CHO cells, the disappearance of blebs, and the formation of stress fibers. We have used immunofluorescence with antibody to chicken gizzard myosin to demonstrate that myosin is present in the blebs of untreated cells and is incorporated into stress fibers following application of db-cAMP. An analysis of the kinetics of myosin redistribution after addition or removal of db-cAMP from CHO cells implies the existence of intermediates in the redistribution of myosin between blebs and stress fibers. In contrast to the rearrangement of myosin, immunofluorescence with antibody to tubulin demonstrates that db-cAMP does not strikingly alter the arrangement of microtubules in CHO cells. Both untreated and db-cAMP-treated cells contain similar arrays of curvilinear microtubules emanating from a perinuclear center. These findings suggest that the establishment of fibroblastic morphology in db-cAMP-treated CHO cells may be a consequence of the formation of myosin-containing stress fibers.