Alterations of catecholamine-sensitive adenylate cyclase in gerbil cerebral cortex after bilateral ischemia

Alterations in central concentrations of cyclic nucleotides occur in mammalian brain during ischemic episodes. Our aim was to evaluate the catecholamine sensitivity of adenylate cyclase using homogenates of the gerbil cerebral cortex during periods of bilateral ischemia and recirculation. After 15-min ischemia without recirculation, the sensitivity of adenylate cyclase was usually enhanced to norepinephrine (NE) and dopamine (DA) alone or in the presence of guanosine triphosphate (GTP) but not 5′-guanylyl imidodiphosphate [Gpp(NH)p]. After 60-min ischemia (no recirculation), the sensitivity of the enzyme to NE and DA showed either no change from sham controls or a slight decrease. Responses, however, in the presence of GTP remained elevated. When the animals were subjected to 15-min ischemia followed by 15-min recirculation, enzyme activation by NE and DA with or without GTP and Gpp(NH)p was usually greater than controls. After 60-min ischemia plus 15-min recirculation, however, enzyme responsiveness to catecholamines and NaF was attenuated. No changes in either high and low K_m cyclic AMP phosphodiesterase or in guanylate cyclase were observed during ischemic episodes. The results indicate that the catecholamine-elicited activity of adenylate cyclase became elevated as a consequence of a short duration of ischemia. Longer periods of ischemia thought to be irreversible showed deficits in adenylate-cyclase sensitivity to catecholamines unless GTP was present. In the presence of GTP, enzyme sensitivity was restored as long as recirculation was prevented. Thus adverse consequences of recirculation on neuronal membranes or receptors could be evaluated by their state of GTP sensitivity.     

beta-adrenergic receptor-adenylate cyclase of denervated sarcolemmal membrane.

Five days after motor denervation, mammalian skeletal muscle sarcolemma undergoes a 50% decline in basal and catecholamine-stimulated adenylate cyclase activity. Sodium fluoride- and 5′-guanylyl imidophosphate [Gpp(NH)p)]-stimulated activities also are depressed. However, properties of the β-adrenergic receptor in the plasma membrane preparation of denervated muscle are similar to those of normal innervated muscle. The number and affinity of specific (?)-[³H]dihydroalprenolol binding sites, the effects of catecholaminergic ligands on binding (isoproterenol > epinephrine > norepinephrine), the stimulatory effects of Gpp(NH)p on adenylate cyclase activity, and the shift in concentration of catecholamines required to activate adenylate cyclase in the presence of Gpp(NH)p were similar in normal and denervated membranes. Thus denervation appears to uncouple the adenylate cyclase response from β-adrenergic stimulation primarily by a loss of adenylate cyclase activity with no change in receptor properties.     

Guanylate cyclase—Cyclic GMP in mouse cerebral cortex and cerebellum: Modification by anticonvulsants

Incubated tissue slices from mouse cerebral cortex and cerebellum readily accumulate cyclic GMP in response to a challenge by ouabain, NaN₃, NH₂OH, or KCl. Under similar conditions, l-glutamate, l-aspartate, glycine, γ-aminobutyric acid, kainic acid, and the calcium ionophore, A-23187, were ineffective. Inhibition of the ouabain-induced accumulation of cyclic GMP was evident with valproate, carbamazepine, clonazepam, phenytoin, and phenobarbital. Only phenytoin blocked the action of KCl and cyclic GMP responses to NaN₃ were inhibited by high concentrations of valproate, carbamazepine, phenytoin, or phenobarbital. The effects of NH₂OH were attenuated by high concentrations of carbamazepine, phenobarbital, and clonazepam (cortex only). Guanylate cyclase activity in homogenates of cortex and cerebellum was enhanced in the presence of NaN₃, NH₂OH, or Ca²⁺ (in low concentrations of Mn²⁺). The enzyme activation induced by Ca⁺ was blocked only by large (1 mm) amounts of carbamazepine. In like manner, large concentrations of carbamazepine, phenytoin (cortex), or clonazepam (cortex) were effective in reducing guanylate cyclase stimulation by NaN₃. No agent affected the NH₂OH responses. The results suggest that anticonvulsant drug actions with regard to central cyclic GMP systems are related to the Na⁺-induced depolarization of nerve tissue and not to any direct actions on the guanylate cyclase enzyme.     

Interactions of antiepileptic drugs on adenylate cyclase and phosphodiesterases in rat and mouse cerebrum.

Five anticonvulsant agents were tested in broken cellular preparations of mouse and rat cerebral cortex for their ability to modify either adenylate cyclase or three forms of cyclic AMP-dependent phosphodiesterase. In both species only carbamazepine inhibited the stimulation of cortical adenylate cyclase by catecholamines (norepinephrine and/or dopamine). Two benzodiazepines, diazepam and clonazepam, enhanced basal and catecholamine-stimulated adenylate cyclase. The latter observations were expecially prominent when phosphodiesterase inhibitors were removed from the preparations. In all instances diazepam was more potent than clonazepam and additionally exerted a more pronounced blockade of high and low K_m phosphodiesterase (mouse cerebrum) and the Ca²⁺-dependent, heatstable, activated phosphodiesterase in the rat cortex. Of the other anticonvulsants evaluated (carbamazepine, phenytoin, and phenobarbital), only phenobarbital inhibited phosphodiesterase, i.e., the low K_m form.     

Adenylate cyclase responses in rat brain after unilateral postnatal X-irradiation

When unilateral x-irradiation was directed to the area of the posterior cerebral cortex overlying the hippocampus of the neonatal rat, histologic damage was observed in the hippocampus (previous observations), pia-arachnoid (cellular proliferation), and retina (two layers of bipolar cells and damage to visual receptor area and diminished number of ganglion cells). Light microscopy did not reveal any remarkable cellular alterations in parenchymal or capillary tissue of the posterior cerebrum. The most prominent deficits in norepinephrine sensitivity of adenylate cyclase occurred in the pia-arachnoid > hippocampus > posterior cerebrum. The associated capillaries were unchanged and in the retina the enzyme displayed an enhanced sensitivity to dopamine.     

Electrophysiologic and cyclic AMP changes in axon-transected frog spinal roots

Transection of a lumbar spinal nerve in the frog produces a disruption of spinal reflexes, a decrease in spinal nerve conduction velocity proximal to the lesion, and alterations in axonal cyclic AMP concentration. Conduction velocity decreases to 85% of control within 7 days of axon transection, and reaches a value 65% of control by 21 days. Monosynaptic spinal reflexes, initiated by either the descending lateral column (LC) pathway or intact lumbar dorsal roots (DRs), show a progressive increase in latency and a decrease in amplitude beginning 17 days postaxotomy. The disruption of reflex pathways continues in nonregenerating systems, but reflexes are restored to normal if regeneration occurs. The predominantly somatic terminations of the LC recover earlier than the predominantly axodendritic synapses of the DR. The signal(s) which initiates these axotomy-induced alterations in neuronal function remains to be identified. The cyclic AMP concentrations of normal and axon-transected spinal roots were measured to determine if cyclic nucleotides could play a role in this communication system. Cyclic AMP increased transiently in spinal roots 6 to 7 days after spinal nerve transection, then returned to control values and eventually began to decline 21 days postaxotomy. With the onset of regeneration, ventral root cyclic AMP concentration returned to control levels, but dorsal root concentration remained depressed. This disassociation between dorsal and ventral roots may reflect a preferential distribution of axonally transported materials into the peripheral process of the sensory axon.     

Effects of dibutyryl cyclic AMP and forskolin on phospholipid biosynthesis in thrombin-stimulated human platelets

The influence of forskolin, an adenylate cyclase activator, and of dibutyryl cyclic AMP (Bt2cAMP) on [3H]glycerol incorporation into glycerolipids was investigated in human platelets. It was found that preincubation with 2.5 mM Bt2cAMP produced a 2-4-fold increase in thrombin-induced incorporation into phospholipids compared to platelets activated by thrombin alone. Pretreatment with forskolin, which increased cellular cAMP content, also resulted in an increase in thrombin-stimulated [3H]glycerol incorporation into phospholipids. These findings demonstrate that a rise in platelet cAMP can accentuate thrombin-induced de novo synthesis of phospholipids from [3H]glycerol. Since the content of cellular cAMP was correlated with its ability to inhibit platelet activation monitored by serotonin release, it seems likely that glycerolipid, in particular phospholipid biosynthesis, is involved in controlling platelet activation by thrombin.     

Schwann cell responses to cyclic AMP: Proliferation, change in shape, and appearance of surface galactocerebroside

Surface galactocerebroside (galC) was induced on cultured Schwann cells by two analogues of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cAMP and 8-bromo cAMP (as previously reported by Sobue and Pleasure) and also by forskolin, a potent adenylate cyclase activator. These reagents also induced a morphological transition of many of the Schwann cells, from an elongated spindle shape to flattened cells extending fenestrated cytoplasmic sheets. Surface galC and these changes in Schwann cell shape were not elicited by raising the extracellular cAMP concentration, nor by many compounds known to promote the differentiation of other cell types, suggesting that intracellular cAMP is the unique signal for their induction. The cAMP analogues also induced Schwann cell proliferation (as previously reported by Raff et al.), as did forskolin. The concentrations of cAMP analogues and forskolin eliciting largest increases in numbers of Schwann cells in the cultures were 10-fold lower than the concentrations required for optimal induction of Schwann cell surface galC.     

Nucleoside triphosphates inhibit ADP,collagen, and epinephrine-induced platelet aggregation: Role of P2Y1 and P2Y12 receptors

Background

Platelets express two ADP receptors namely P2Y₁ and P2Y₁₂ that regulate ADP and other agonists-induced platelet aggregation. P2Y₁ receptor activation causes platelet shape change while P2Y₁₂ receptor activation induces platelet aggregation. Previously, anti-aggregatory effects of ATP on ADP-induced and pro-aggregatory effects on epinephrine-induced platelet aggregation have been reported. However, the effects of other nucleoside triphosphates on platelet aggregation have never been described. The aim of the present study was to characterise the effects of nucleoside triphosphates (ATP, UTP, GTP, and CTP) on agonist-induced platelet aggregation.

Methods

The experiments were performed on platelet rich plasma freshly isolated from blood donated by healthy human volunteers.

Results

All the nucleoside triphosphates tested inhibited ADP- and collagen-induced platelet aggregation in a concentration-dependent manner with a rank order of potency, 2MeSATP > ATP ≥ α,β,methyleneATP > UTP  >> CTP ≥ GTP. The IC₅₀ values against ADP (10 μM)-induced platelet aggregation were 0.039 ± 0.013, 18 ± 7, 25 ± 6, 32 ± 9, 360 ± 130, and 400 ± 160 μM, respectively. Low concentrations of ATP induced platelet shape change which was due to contaminating ADP. However, higher concentrations antagonised ADP and MRS2365-induced platelet shape change. The ATP analogue α,β,methyleneATP and CTP but not UTP and GTP also antagonised ADP-induced platelet shape change. Similarly, low ATP concentrations potentiated epinephrine-induced platelet aggregation that was abolished by P2Y₁ antagonist MRS2500 suggesting P2Y₁ receptor activation due to contaminating ADP. Higher ATP concentrations, α,β,methyleneATP, UTP, CTP, and GTP antagonised epinephrine-induced platelet aggregation.

Conclusion

Thus, the data demonstrate nucleoside triphosphates in general act as P2Y₁₂ receptor antagonists and antagonise ADP-, collagen-, and epinephrine-induced platelet aggregation.     

Changes in energy metabolites, cGMP and intracellular pH during cortical spreading depression

The spatial and temporal distribution of cerebral metabolites and pH_i were examined in the cortex during spreading depression. Acidification and marked depression in the energy status of the tissue was evident at the wavefront of spreading depression. In its aftermath, the residual activation of glycolysis and accumulation of cGMP persisted for minutes after a relatively rapid restoration of high-energy phosphates and pH_i.     

Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons

Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3)) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP(3) receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP(3) in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.     

Uliginosin B presents antinociceptive effect mediated by dopaminergic and opioid systems in mice

Previous studies have shown that uliginosin B inhibits dopamine reuptake in rat brain. This compound occurs in Hypericum polyanthemum and H. caprifoliatum for which was reported to have antinociceptive effect sensitive to naloxone. The aim of this study was to assess the antinociceptive effect of uliginosin B and to evaluate the involvement of opioid and dopaminergic receptors activation. Uliginosin B presented antinociceptive effect in hot-plate and abdominal writhing tests, in mice, at doses that did not impair the motor coordination (15 mg/kg, i.p.). Uliginosin B in high dose (90 mg/kg, i.p.) presented ataxic effect in the rotarod apparatus. These effects seem to be mediated by distinct receptors since the effect on the hot-plate was completely abolished by naloxone and sulpiride, but it was unaffected by SCH 23390. On the other hand, the motor impairment induced by uliginosin B was completely prevented by naloxone and partially prevented by sulpiride and SCH 23390. However, the receptors' activation appears to be indirect since uliginosin B did not bind to opioid and dopaminergic receptors. Thus, uliginosin B effects probably are due to its ability to inhibit monoamine reuptake with consequent activation of dopamine receptors and indirect stimulation of opioid system.     

Dopamine D2 receptor partial agonists display differential or contrasting characteristics in membrane and cell-based assays of dopamine D2 receptor signaling

Clinical evidence suggests that dopamine D(2) receptor partial agonists must have a sufficiently low intrinsic activity to be effective as antipsychotics. Here, we used dopamine D(2) receptor signaling assays to compare the in vitro functional characteristics of the antipsychotic aripiprazole with other dopamine D(2) receptor partial agonists (7-{3-[4-(2,3-dimethylphenyl)-piperazinyl]propoxy}-2(1H)-quinolinone [OPC-4392], (-)-3-(3-hydroxy-phenyl)-N-n-propylpiperidine [(-)3-PPP] and (+)terguride) and dopamine D(2) receptor antagonists. Aripiprazole and OPC-4392 were inactive in a guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding assay using Chinese Hamster Ovary (CHO) cell membranes expressing cloned human dopamine D(2Long) (hD(2L)) receptors, whereas (-)3-PPP and (+)terguride displayed low intrinsic activity. Aripiprazole also had no effect on [(35)S]GTPgammaS binding to CHO-hD(2L) cells, while OPC-4392, (-)3-PPP and (+)terguride were partial agonists. In contrast, aripiprazole, OPC-4392, (-)3-PPP, and (+)terguride were inactive in a [(35)S]GTPgammaS binding assay using rat striatal membranes. However, at a more downstream level of CHO-hD(2L) cell signalling, these drugs all behaved as dopamine hD(2L) receptor partial agonists, with aripiprazole displaying an intrinsic activity 2 to 3-fold lower (inhibition of forskolin-induced adenosine 3',5'-cyclic monophosphate accumulation) and almost half as high (enhancement of adenosine triphosphate-stimulated [(3)H]arachidonic acid release) as OPC-4392, (-)3-PPP and (+)terguride. Dopamine activity was blocked in each case by (-)raclopride, which was inactive on its own in every assay, as were the antipsychotics haloperidol, olanzapine, ziprasidone and clozapine. Together, these data, whilst preclinical in nature, are consistent with clinical evidence suggesting the favorable antipsychotic profile of aripiprazole, compared with the other clinically ineffective partial agonists, is dependent on its low intrinsic activity at dopamine D(2) receptors. This study also highlights the limitations of using [(35)S]GTPgammaS binding assays to identify dopamine D(2) receptor partial agonists.     

Low affinity binding of the classical D1 antagonist SCH23390 in rodent brain: potential interaction with A2A and D2-like receptors

Whereas structurally dissimilar D(1) antagonists competing for [(3)H]-SCH23390 binding recognize primarily one site in striatum, two distinct affinity states are observed in both amygdala and hippocampus. The binding profile of SCH23390 is similar in both of these regions, with the high affinity site (K(D) approximately 0.4 nM) consistent with D(1)/D(5) receptors. The appearance of the low affinity site (K(D) approximately 300 nM) is dependent upon the absence of MgCl(2), but independent of D(1) expression (i.e., still present in D(1) knockout mice). Although the density of high affinity state receptor is lower in hippocampus or amygdala of D(1) knockout mice, some residual binding remains, consistent with the known expression of D(5) receptors in these regions. Remarkably, in hippocampus, the affinity of the low affinity site is shifted rightward in the presence of the D(2) antagonist domperidone and is largely absent in the hippocampus of D(2) knockout animals. Additionally, this site is also shifted rightward in the presence of the A(2A) ligands SCH58261, CSC, or NECA, or in the absence of A(2A) receptors. The affinity of SCH23390 for this low affinity site is greater than seen for SCH23390 binding to D(2) receptors in heterologous expression systems, consistent with the hypothesis that both D(2) and A(2A) receptors are involved in the low affinity binding site. Therefore, we suggest that the heteromerization of D(2) and A(2A) receptors reported previously in vitro also may occur in the brain of both rats and mice.     

Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I_h). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I_h properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I_h was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I_h found in WAG/Rij rats compensate each other in a way that leaves I_h availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I_h on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I_h in older animals.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

Somatostatin receptors: From signaling to clinical practice

Somatostatin is a peptide with a potent and broad antisecretory action, which makes it an invaluable drug target for the pharmacological management of pituitary adenomas and neuroendocrine tumors. Somatostatin receptors (SSTR1, 2A and B, 3, 4 and 5) belong to the G protein coupled receptor family and have a wide expression pattern in both normal tissues and solid tumors. Investigating the function of each SSTR in several tumor types has provided a wealth of information about the common but also distinct signaling cascades that suppress tumor cell proliferation, survival and angiogenesis. This provided the rationale for developing multireceptor-targeted somatostatin analogs and combination therapies with signaling-targeted agents such as inhibitors of the mammalian (or mechanistic) target of rapamycin (mTOR). The ability of SSTR to internalize and the development of rabiolabeled somatostatin analogs have improved the diagnosis and treatment of neuroendocrine tumors.     

Molecular regulation of dendritic spine dynamics and their potential impact on synaptic plasticity and neurological diseases

The structure and dynamics of dendritic spines reflect the strength of synapses, which are severely affected in different brain diseases. Therefore, understanding the ultra-structure, molecular signaling mechanism(s) regulating dendritic spine dynamics is crucial. Although, since last century, dynamics of spine have been explored by several investigators in different neurological diseases, but despite countless efforts, a comprehensive understanding of the fundamental etiology and molecular signaling pathways involved in spine pathology is lacking. The purpose of this review is to provide a contextual framework of our current understanding of the molecular mechanisms of dendritic spine signaling, as well as their potential impact on different neurodegenerative and psychiatric diseases, as a format for highlighting some commonalities in function, as well as providing a format for new insights and perspectives into this critical area of research. Additionally, the potential strategies to restore spine structure–function in different diseases are also pointed out. Overall, these informations should help researchers to design new drugs to restore the structure–function of dendritic spine, a “hot site” of synaptic plasticity.