Withdrawal from butorphanol dependence alters binding of [H]phorbol dibutyrate to protein kinase C, but not of [H]forskolin to adenylate cyclase

The time course of autoradiographic binding of major second messengers in the rat brain was studied at 2, 7, and 24 h after withdrawal from butorphanol infusion. [³H]Forskolin and [³H]phorbol 12,13-dibutyrate (PDBu) were used to label adenylate cyclase and protein kinase C (PKC), respectively. Rats were rendered dependent by intracerebroventricular infusion of butorphanol (26 nmol μl⁻¹ h⁻¹) via osmotic minipumps for 3 days. Withdrawal was initiated by abrupt cessation of the butorphanol infusion. The levels of [³H]forskolin binding were not changed at any time or in any brain area, except for an increase following 7 h of withdrawal in the brainstem only. The levels of [³H]PDBu binding were significantly increased (13–47%) in multiple areas of the rat brain following 7 h of withdrawal from butorphanol infusion. These findings suggest that the phosphoinositide cycle system is more susceptible to alteration during butorphanol dependence than is the adenylate cyclase system in the rat brain.     

Odor increases [3H]phorbol dibutyrate binding to protein kinase C in olfactory structures of rat brain. Effect of entorhinal cortex lesion

Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.     

Inhibition of [3H]forskolin binding by Gpp[NH]p may reflect adenylate cyclase coupling to Gi.

The effect of the GTP analogue guanylyl-5'-imidodiphosphate (Gpp[NH]p) on [3H]forskolin binding was studied in rat brain using autoradiography. In the striatum the presence of 100 microM Gpp[NH]p produced a 40% increase in binding, whereas a decrease of about 30% was observed with low Gpp[NH]p concentrations (0.1 microM). In the molecular layer of the cerebellum all concentrations of Gpp[NH]p decreased [3H]forskolin binding. The decrease in binding disappeared in both striatum and the molecular layer of cerebellum in sections pretreated with 100 microM N-ethylmaleimide (NEM) for 10 min. NEM pretreatment did not significantly affect the stimulation of [3H]forskolin binding by micromolar concentrations of Gpp[NH]p in the striatum, but reversed the decrease observed in the molecular layer of the cerebellum, to an increase. Based on these data we suggest that the effects of Gpp[NH]p on [3H]forskolin binding may involve both Gs and Gi, where stimulation produces an increase and decrease in binding, respectively. The regional effects of Gpp[NH]p may reflect differences in the responsiveness of adenylate cyclase to Gs- and Gi-mediated effects.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

[H]5-HT binding sites and 5-HT-sensitive adenylate cyclase in glial cell membrane fraction

Glial cell membrane fractions were prepared using glial cells preparations isolated from horse brain striatum. [³H]5-HT binding was measured by the filtration technique and the adenylate cyclase activity determined by measuring the cAMP production using a radioimmunoassay.Serotonin binds to glial membrane fractions with an affinity corresponding to a dissociation constantK_d = 10 nM. The corresponding site is serotoninergic specific: [³H]5-HT binding is inhibited by 5-HT agonists (5 OH NM-DMT, 5-MeOHT, 5-MeOH-DMT, NN-DMT) or antagonists (cinanserine, cyproheptadine, methysergide, LSD) and not (or poorly) inhibited by non-serotoninergic related drugs. The population of sites binding 5-HT, present in neuronal membrane preparations and determined in parallel assays is distinct from that observed in glial preparations. The glial membrane fractions contains an adenylate cyclase activated by 5-HT with an apparent affinity constant close to 1 μM. It is serotonin-specific and clearly distinct from the DA-stimulated adenylate cyclase present in the same preparation.The sites binding 5-HT and activating the adenylate cyclase with low affinities might be directly related. This system, clearly distinct from the postsynaptosomal serotoninergic receptor, represents presumably a glial serotoninergic receptor; however, it cannot be totally excluded that these sites may refer to presynaptic membranes.     

Alteration of protein kinase C activity in the postischemic rat brain areas using in vitro [3H]phorbol 12,13-dibutyrate autoradiography

Summary Chronological changes of protein kinase C (PKC) activity were measured using in vitro [³H]phorbol 12,13-dibutyrate (PDBu) autoradiography to investigate the postischemic alteration of this second messenger system in the rat brain. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 hours of recirculation, [³H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [³H]PDBu binding sites was first detected in the ipsilateral thalamus and the substantia nigra, which both areas had not been directly affected by the original ischemic insult. This postischemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with⁴⁵Ca accumulation, which was detected there in our previous study. These results suggest that alteration of second messenger (PKC) pathways may be involved not only in the ischemic foci, but also in neuronal degeneration of the exo-focal remote areas in relation to the disruption of intracellular calcium homeostasis which plays a key role in the pathogenesis of postischemic neuronal damage and that marked alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.     

Autoradiographic study of NMDA-displaceable [3H]glutamate and [3H]MK-801 binding during butorphanol withdrawal in the rat brain.

Influences of continuous administration of butorphanol on the autoradiography of [3H]glutamate binding and [3H]MK-801 binding were investigated to study the effects of butorphanol withdrawal on NMDA receptors. Rats were administered butorphanol (26 nmol microl(-1) h(-1)) by continuous intracerebroventricular (i.c.v.) infusion through pre-implanted cannula connected to osmotic mini-pumps for 3 days. Rats were then sacrificed at 2, 7, and 24 h after discontinuation of butorphanol infusion. [3H]MK-801 binding was slightly increased in the cortical area, hippocampus, and cerebellum in 2, 7, and 24 h withdrawal groups and was shown most significant increase in the 7 h withdrawal group. NMDA-displaceable [3H]glutamate binding was markedly increased in the cortical area, striatum, septum, hippocampus, thalamus, and cerebellum in 7 h withdrawal group and was significantly increased in the striatum, hippocampus, and thalamus in 24 h withdrawal group. These results demonstrate that the development of butorphanol withdrawal is more prominent by 7 h after discontinuation of butorphanol infusion and suggest that NMDA binding sites at NMDA receptors may play more important role in the development of butorphanol withdrawal than that of channel blocking sites.     

Dissociation between the presynaptic dopamine-sensitive adenylate cyclase and [H]spiperone binding sites in rat substantia nigra

[3H]Spiperone binding sites and the dopamine-sensitive adenylate cyclase were measured in rat substantia nigra (s. nigra) 7 or 14 days after various lesions. Hemisections, which resulted in a 66% decline in tyrosine hydroxylase and cyclic nucleotide phosphodiesterase and a 73% decrease in glutamate decarboxylase, led to a 50% decrease in [3H]spiperone binding and to the almost complete disappearance of the dopamine-sensitive adenylate cyclase from the s. nigra on the lesioned side. 6-Hydroxydopamine injection into the s. nigra, which depleted tyrosine hydroxylase activity within the s. nigra by 85%, while leaving phosphodiesterase unaffected, resulted in a 40% decrease in [3H]spiperone binding but no change in the dopamine-sensitive adenylate cyclase. Intrastriatal injections of kainic acid did not alter tyrosine hydroxylase activity in the s. nigra, but decreased both glutamate decarboxylase (54%) and phosphodiesterase (68%); [3H]spiperone binding was unaffected by this lesion while the dopamine-sensitive adenylate cyclase was greatly reduced (50-75%). These results suggest that within the s. nigra the dopamine receptor binding sites as defined using [3H]spiperone are located on dopamine neurones while the dopamine-sensitive adenylate cyclase is located presynaptically on striatonigral nerve terminals.     

Quantitative autoradiography of [H]forskolin binding sites in the rat brain

The binding sites for a radiolabeled form of the potent activator of adenylate cyclase, forskolin, have been localized in the rat brain, pituitary and spinal cord. Using the quantitative technique of in vitro autoradiography, a high density of [3H]forskolin binding was detected in brain structures such as the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra and the hilus of the area dentata. A comparison of the distribution of [3H] forskolin binding sites with those reported for several neurotransmitter receptor types indicated that forskolin identified adenylate cyclase was probably not linked to any single type of neurotransmitter receptor. These results also presented several new brain areas in which to investigate the neuronal role of adenylate cyclase.     

Platelet P-selectin expression: requirement for protein kinase C,but not protein tyrosine kinase or phosphoinositide 3-kinase

P-selectin is translocated from the alpha-granules to the surface of activated platelets where it participates in thrombosis and inflammation. We investigated the signaling pathways involved in thrombin-induced human platelet P-selectin expression. Assessed by flow cytometry, inhibition of protein kinase C (PKC) with chelerythrine reduced P-selectin expression by 66%, platelet/neutrophil binding, GPIIb/IIIa activation and aggregation (p<0.05). G? 6976, an inhibitor of the conventional PKCs (alpha and beta), did not alter P-selectin expression. However, rottlerin inhibited by 50% its expression (p<0.05), but only at doses that interfere with the novel (epsilon eta) and atypical (zeta) PKCs. Inhibition of protein tyrosine kinase (PTK) and phosphoinosi-tide 3-kinase (PI3-K) did not significantly affect P-selectin expression. In conclusion, thrombin-induced P-selectin expression is PKC-sensitive, but PTK and PI3-K-insensitive. The novel epsilon and eta and atypical zeta, but not the conventional alpha and beta and the novel delta PKCs, may be involved in this process.     

Activation of protein kinase C by phorbol dibutyrate potentiates [

Using phorbol 12,13-dibutyrate (PDBu) autoradiography, we investigated the effect of hypothermia on protein kinase C (PKC) activation in rat brain 2 h after focal ischemia. In normothermia, a significant increase of PDBu binding was observed in ischemic brain. Hypothermia suppressed the increase of PDBu binding in degree and extent. These observations suggest that intraischemic hypothermia attenuates the activation of PKC, and this may in part be participate in the protective effect of hypothermia.     

Hypothermia attenuates the activation of protein kinase C in focal ischemic rat brain: dual autoradiographic study of [H]phorbol 12,13-dibutyrate and iodo[C]antipyrine

Using phorbol 12,13-dibutyrate (PDBu) autoradiography, we investigated the effect of hypothermia on protein kinase C (PKC) activation in rat brain 2 h after focal ischemia. In normothermia, a significant increase of PDBu binding was observed in ischemic brain. Hypothermia suppressed the increase of PDBu binding in degree and extent. These observations suggest that intraischemic hypothermia attenuates the activation of PKC, and this may in part be participate in the protective effect of hypothermia.     

Post- and presynaptic lesions in the CA1 region of hippocampus: Effect on [3H]forskolin and [3H]phorboldibutyrate ester binding

Summary The effect of transient cerebral ischemia and intraventricular injection of kainic acid on adenylate cyclase and protein kinase C as labeled by [³H]forskolin ([³H]FOR) and [³H]phorboldibutyrate ester ([³H]PDBU) in several rat brain microregions was investigated in a quantitative autoradiographic study. Four days after transient four vessel occlusion a 80% loss of [³H]FOR and a 35% loss of [³H]PDBU binding could be measured in the CA1 stratum radiatum of operated Wistar rats as compared to control rats. Four days after intraventricular injection of kainic acid only a marginal loss of [³H]FOR and a 30% increase of [³H]PDBU binding was seen in the CA1 stratum radiatum while in the CA3 stratum lucidum and radiatum respectively a 30% loss of [³H]FOR and no significant change in [³H]PDBU binding was observed. As transient cerebral ischemia and intraventricular kainic acid injection are depleting the hippocampal CA1 region of CA1 pyramidal cells and axons of CA3 pyramidal cells respectively in rat brain, these findings strongly suggest that both adenylate cyclase and protein kinase C are localized in CA1 pyramidal cells of rat hippocampus.Part of this study has been presented at the 16th C.I.N.P. Congress, Munich, August 15–19, 1988.     

Effects of long-term treatment with lithium and antidepressants on [3H]forskolin binding to rat cerebral cortical membranes

1. The characteristics of [3H]forskolin binding in rat cerebral cortical membranes and the effects of long-term treatment with lithium, desipramine and tranylcypromine on the binding were investigated. 2. Specific binding of [3H]forskolin (10 nM) was augmented by the addition of magnesium, and in the presence of 5 mM MgCl2, sodium fluoride (NaF) increased further the specific binding in a concentration-dependent manner. 3. Guanyl-5'-ylimidodiphosphate (Gpp(NH)p) increased the specific binding only in the presence of 100 mM MgCl2. 4. Long-term treatment with lithium, desipramine or tranylcypromine altered neither the specific binding of [3H]forskolin nor the augmentation by MgCl2, NaF, or Gpp(NH)p. 5. The results indicate that these preparations alter neither the catalytic subunit of adenylate cyclase nor the activation of stimulatory guanine nucleotide-binding regulatory proteins (Gs).     

Decreased phorbol ester binding in the parahippocampal gyrus from subjects with schizophrenia is not associated with changes in protein kinase C

Combining in situ radioligand binding with autoradiography, we previously identified a reduction of [(3)H]phorbol 12,13-dibutyrate binding in the parahippocampal gyrus from schizophrenic subjects. To determine whether these changes were due to decreases in the level of protein kinase C, we measured [(3)H]phorbol 12,13-dibutyrate binding, levels of the protein kinase C isoforms alpha, beta, delta, epsilon, gamma, eta and theta, as well as protein kinase C activity in crude particulate membranes from parahippocampal gyri of 15 schizophrenic and 15 control subjects. There was a significant decrease in the density (mean +/- SEM: 6.56 +/- 0.73 pmol mg(-1) vs 9.68 +/- 1.22 pmol mg(-1); P < 0.05) and affinity (mean K(D) +/- SEM: 4.64 +/- 0.34 nM vs 2.95 +/- 0.35 nM; P < 0.005) of [(3)H]phorbol 12,13-dibutyrate binding in homogenates from schizophrenic subjects. There were no significant changes in levels of the protein kinase C isoforms which are known to bind phorbol esters or in the activity of protein kinase C in membranes from schizophrenic subjects. These results suggest that there are changes in molecules capable of binding [(3)H]phorbol 12,13-dibutyrate, other than protein kinase C, in the parahippocampal gyrus from subjects with schizophrenia.