Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions.

Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5 µg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain.

Role of ethanolamine phosphate in the hippocampus of rats with acute experimental autoimmune encephalomyelitis.

Here, we assessed the effects of acute experimental autoimmune encephalomyelitis (EAE) on the rat hippocampal somatostatinergic system and whether administration of an ethanolamine phosphate salt could prevent the appearance of the clinical signs and the impairment of the somatostatinergic system in this pathological condition. Female Lewis rats were injected in both hindlimb footpads with myelin basic protein from guinea pig brain and complete Freund's adjuvant and were sacrificed when limp tail (grade 1 EAE) or severe hindlimb paralysis (grade 3 EAE) were observed. One group was injected daily with ethanolamine phosphate, starting two days prior to immunization and for 15 days thereafter. The animals were sacrificed 15 days post-immunization. Acute EAE in grade 3 increased anti-myelin basic protein antibodies in rat serum as well as tumor necrosis factor-α and interferon-γ levels in hippocampal extracts. In addition, it decreased the somatostatin receptor density, somatostatin receptor subtype 2 mRNA and protein content, and the inhibitory effect of somatostatin on adenylyl cyclase activity in the hippocampus. The protein levels of the inhibitory G protein subunits αi(1-3), the G protein-coupled receptor kinase isoforms 2, 5 and 6, the phosphorylated cyclic AMP-binding protein and the somatostatin-like immunoreactivity content were unaltered in this brain area. Acute EAE in grade 1 did not modify any of these parameters. Ethanolamine phosphate administration prevented the clinical expression of acute EAE as well as the decrease in the somatostatin receptor density, somatostatin receptor subtype 2 expression and the capacity of somatostatin to inhibit adenylyl cyclase activity at the time-period studied. Furthermore, it blunted the rise in serum anti-myelin basic protein antibodies and hippocampal interferon-γ and tumor necrosis factor-α levels. Altogether, these data suggest that ethanolamine phosphate might provide protection against acute EAE.

alpha-Tocopherol decreases the somatostatin receptor-effector system and increases the cyclic AMP/cyclic AMP response element binding protein pathway in the rat dentate gyrus.

Neuronal survival has been shown to be enhanced by alpha-tocopherol and modulated by cyclic AMP (cAMP). Somatostatin (SST) receptors couple negatively to adenylyl cyclase (AC), thus leading to decreased cAMP levels. Whether alpha-tocopherol can stimulate neuronal survival via regulation of the somatostatinergic system, however, is unknown. The aim of this study was to investigate the effects of alpha-tocopherol on the SST signaling pathway in the rat dentate gyrus. To that end, 15-week-old male Sprague-Dawley rats were treated daily for 1 week with (+)-alpha-tocopherol or vehicle and sacrificed on the day following the last administration. No changes in either SST-like immunoreactivity (SST-LI) content or SST mRNA levels were detected in the dentate gyrus as a result of alpha-tocopherol treatment. A significant decrease in the density of the SST binding sites and an increase in the dissociation constant, however, were detected. The lower SST receptor density in the alpha-tocopherol-treated rats correlated with a significant decrease in the protein levels of the SST receptor subtypes SSTR1-SSTR4, whereas the corresponding mRNA levels were unaltered. G-protein-coupled-receptor kinase 2 expression was decreased by alpha-tocopherol treatment. This vitamin induced a significant increase in both basal and forskolin-stimulated AC activity, as well as a decrease in the inhibitory effect of SST on AC. Whereas the protein levels of AC type V/VI were not modified by alpha-tocopherol administration, ACVIII expression was significantly enhanced, suggesting it might account for the increase in AC activity. In addition, this treatment led to a reduction in Gialpha1-3 protein levels and in Gi functionality. alpha-Tocopherol did not affect the expression of the regulator of G-protein signaling 6/7 (RGS6/7). Finally, alpha-tocopherol induced an increase in the levels of phosphorylated cAMP response element binding protein (p-CREB) and total CREB in the dentate gyrus. Since CREB synthesis and phosphorylation promote the survival of many cells, including neurons, whereas SST inhibits the cAMP-PKA pathway, which is known to be involved in CREB phosphorylation, the alpha-tocopherol-induced reduction of SSTR observed here might possibly contribute, via increased cAMP levels and CREB activity, to the mechanism by which this vitamin promotes the survival of newborn neurons in the dentate gyrus.

Sulfadiazine partially protects the rat temporal cortex from amyloid beta peptide (25-35)-induced alterations of the somatostatinergic system.

The aim of this study was to elucidate whether sulfadiazine, shown to improve cognitive capacity in the elderly, can prevent amyloid beta peptide (Abeta) (25-35)-induced impairment of the somatostatinergic system previously reported by our group in rat temporal cortex. Male Wistar rats were thus treated with sulfadiazine (160 mg/kg) or vehicle, via a gastric cannula, twice on the day prior to Abeta(25-35) treatment. On the following day and during 14 days, Abeta(25-35) was administered intracerebroventricularly (i.c.v.) via an osmotic minipump connected to a cannula implanted in the right lateral ventricle (300 pmol/day). Sulfadiazine (80 mg/kg) or vehicle was administered once again the last 2 days of the Abeta(25-35) infusion. All animals were sacrificed by decapitation 24 h after the last sulfadiazine dose. The findings obtained reveal that sulfadiazine partially prevents the decrease in somatostatin (SRIH)-like immunoreactivity content in the temporal cortex of rats infused with Abeta(25-35) during 14 days. In addition, sulfadiazine blocks the Abeta(25-35)-induced reduction in the SRIH receptor density and in SRIH receptor subtype 2 expression. Sulfadiazine treatment also restored the inhibitory effect of SRIH on basal adenylyl cyclase activity back to control values. Altogether, the results suggest that sulfadiazine might have beneficial effects in the early treatment of Alzheimer's disease.

Minocycline prevents Abeta(25-35)-induced reduction of somatostatin and neprilysin content in rat temporal cortex.

AIMS: Tetracyclines have been demonstrated to inhibit formation of beta-amyloid (Abeta) aggregates and to disassemble preformed fibrils. Minocycline, a semi-synthetic second-generation tetracycline, can reverse Abeta-induced impairment of cognitive functions. Since somatostatin is involved in cognition and we recently showed that Abeta(25-35) lowers somatostatin expression in the rat temporal cortex, our aim here was to analyze the effects of minocycline on somatostatin immunoreactivity and mRNA levels in the temporal cortex of Abeta(25-35)-infused and healthy rats. Moreover, since brain levels of neprilysin, an Abeta-degrading enzyme, decrease with age, favoring the appearance of senile neuritic plaques, we tested whether minocyline could affect neprilysin expression. MAIN METHODS: Wistar rats were thus injected with minocycline twice on the first day of treatment. On the following day, and during 14 days, Abeta(25-35) or vehicle were administered. Minocycline was injected once again on days 13 and 14. All animals were sacrificed 24 h after the last drug injection. KEY FINDINGS: Minocycline abrogated the Abeta(25-35)-induced decrease of somatostatin-like immunoreactive content, somatostatin mRNA levels, phosphorylated-CREB content and neprilysin levels. Minocycline alone enhanced these targets. SIGNIFICANCE: Our findings indicate that minocycline prevents the deleterious effects of Abeta(25-35) on SRIF and neprilysin expression in the rat temporal cortex and that it has protective effects per se on these parameters.

Minocycline provides protection against beta-amyloid(25-35)-induced alterations of the somatostatin signaling pathway in the rat temporal cortex.

Minocycline is a semi-synthetic second-generation tetracycline known to improve cognition in amyloid precursor protein transgenic mice. Whether it can protect the somatostatin (SRIF) receptor-effector system, also involved in learning and memory, from alterations induced by chronic i.c.v. infusion of beta-amyloid peptide (Abeta)(25-35) is presently unknown. Hence, in the present study, we tested the effects of minocycline on the SRIF signaling pathway in the rat temporal cortex. To this end, male Wistar rats were injected with minocycline (45 mg/kg body weight) i.p. twice on the first day of treatment. On the following day and during 14 days, Abeta(25-35) was administered i.c.v. via an osmotic minipump connected to a cannula implanted in the left lateral ventricle (300 pmol/day). Minocycline (22.5 mg/kg, i.p.) was injected once again the last 2 days of the Abeta(25-35) infusion. The animals were killed by decapitation 24 h after the last drug injection. Our results show that minocycline prevents the decrease in SRIF receptor density and somatostatin receptor (sst) 2 expression and the attenuated capacity of SRIF to inhibit adenylyl cyclase (AC) activity, alterations present in the temporal cortex of Abeta(25-35)-treated rats. Furthermore, minocycline blocks the Abeta(25-35)-induced decrease in phosphorylated cyclic AMP (cAMP) response element binding protein (p-CREB) content and G-protein-coupled receptor kinase 2 (GRK) protein expression in this brain area. Altogether, the present data demonstrate that minocycline in vivo provides protection against Abeta-induced impairment of the SRIF signal transduction pathway in the rat temporal cortex and suggest that it may have a potential as a therapeutic agent in human Alzheimer's disease, although further studies are warranted.

Somatostatin and Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the cerebral deposition of senile plaques that are mainly composed of a set of peptides referred to as amyloid beta-peptides (Abeta). Among the numerous neuropeptides produced in intrinsic cortical and hippocampal neurons, somatostatin (SRIF) has been found to be the most consistently reduced in the brain and cerebrospinal fluid of AD patients. SRIF receptors (SSTR), which mediate the neuromodulatory signals of SRIF, are also markedly depleted in the AD brain, there being subtype-selective alterations in cortical areas. In the rat temporal cortex, we have shown that intracerebroventricular infusion of Abeta25-35 results in a decrease in SRIF-like immunoreactivity and in SRIF receptor subtype 2 (SSTR2) mRNA and protein levels, in correlation with a decrease in SSTR functionality. Insulin-like growth factor-I prevents the reduction in these parameters induced by Abeta25-35. Abeta has recently been demonstrated to be degraded primarily by a neutral endopeptidase, neprilysin, in the brain. SRIF regulates brain Abeta levels via modulation of neprilysin activity. Because SRIF expression in the brain declines upon aging in various mammals, including rodents, apes and humans, the aging-dependent reduction of SRIF has been hypothesized to trigger accumulation of Abeta in the brain by suppressing neprilysin action. Here we present an overview of recent advances on the role of SRIF in AD and its relationship with Abeta peptides.

Alteration of the somatostatinergic system in the striatum of rats with acute experimental autoimmune encephalomyelitis.

To date, the neurochemical basis underlying the motor and cognitive deficits described in patients with multiple sclerosis (MS) is unclear. Since the neuropeptide somatostatin (SRIF) and the striatum have been implicated in movement control and implicit memory, the aim of this study was to analyze the striatal somatostatinergic system in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). Female Lewis rats were immunized with an emulsion containing myelin basic protein (MBP) in complete Freund's adjuvant to induce the disease. The animals were decapitated when limp tail (grade 1) or severe hind limb paralysis (grade 3) was observed. Acute EAE in grade 3 did not modify striatal somatostatin-like immunoreactivity (SRIF-LI) content but decreased the overall SRIF receptor density, without affecting the apparent affinity, in the rat striatal membranes. A selective reduction in the protein levels of the SRIF receptor subtype sst2, analyzed by Western blotting, was detected in the EAE rats, which correlated with decreased sst2 mRNA levels. The expression of the receptor subtypes sst1, sst3 or sst4 was unaltered by the disease. The decrease in the SRIF receptor density was accompanied by an attenuated capacity of SRIF to inhibit both basal and forskolin-stimulated adenylyl cyclase activity. No significant changes, however, were found in the protein levels of Gi proteins (G(ialpha1), G(ialpha2) or G(ialpha3)) nor in those of the G-protein-coupled receptor kinase subtypes GRK2, GRK5 or GRK6. Acute EAE in grade 1 did not modify any of the parameters studied. In conclusion, these data demonstrate that acute EAE, in grade 3, disrupts the rat striatal SRIF receptor-effector system. These findings provide new insight into the molecular basis of EAE which might contribute to a better understanding of multiple sclerosis in humans.

Chronic but not acute intracerebroventricular administration of amyloid beta-peptide(25-35) decreases somatostatin content, adenylate cyclase activity, somatostatin-induced inhibition of adenylate cyclase activity, and adenylate cyclase I levels in the rat hippocampus.

Although alterations in adenylate cyclase (AC) activity and somatostatin (SRIF) receptor density have been reported in Alzheimer's disease, the effects of amyloid beta-peptide (Abeta) on these parameters in the hippocampus are unknown. Our aim was to investigate whether the peptide fragment Abeta(25-35) can affect the somatostatinergic system in the rat hippocampus. Hence, Abeta(25-35) was injected intracerebroventricularly (i.c.v.) to Wistar rats in a single dose or infused via an osmotic minipump connected to a cannula implanted in the right lateral ventricle during 14 days. The animals were decapitated 7 or 14 days after the single injection and 14 days after chronic infusion of the peptide. Chronic i.c.v. infusion of Abeta(25-35) decreased SRIF-like immunoreactive content without modifying the SRIF receptor density, SRIF receptor expression, or the Gialpha(1), Gialpha(2), and Gialpha(3) protein levels in the hippocampus. This treatment, however, caused a decrease in basal and forskolin-stimulated AC activity as well as in the capacity of SRIF to inhibit AC activity. Furthermore, the protein levels of the neural-specific AC type I were significantly decreased in the hippocampus of the treated rats, whereas an increase in the levels of AC V/VI was found, with no alterations in type VIII AC. A single i.c.v. dose of Abeta(25-35) exerted no effect on SRIF content or SRIF receptors but induced a slight decrease in forskolin-stimulated AC activity and its inhibition by SRIF. Because chronic Abeta(25-35) infusion impairs learning and memory whereas SRIF facilitates these functions, the alterations described here might be physiologically important given the decreased cognitive behavior previously reported in Abeta-treated rats.

A vitamin A-free diet results in impairment of the rat hippocampal somatostatinergic system.

Previous studies have revealed the presence of retinoid specific receptors in the hippocampus and have demonstrated that vitamin A deficiency produces a severe deficit in spatial learning and memory which are linked to a proper hippocampal functioning. It is also well known that the tetradecapeptide somatostatin binds to specific receptors in the hippocampus and, when injected into this brain area, facilitates the acquisition of spatial tasks. In addition, depletion of somatostatin by cysteamine impairs acquisition of these tasks. Taken together, these studies support the idea that the hippocampal somatostatinergic system might be regulated by vitamin A. Hence, we evaluated the effects of vitamin A deprivation and subsequent administration of vitamin A on the rat hippocampal somatostatinergic system. Rats fed a vitamin A-free diet exhibited a significant reduction of somatostatin-like immunoreactivity content in the hippocampus whereas the somatostatin mRNA levels were unaltered. Vitamin A deficiency increased the somatostatin receptor density and its dissociation constant. Functional Gi activity as well as the capacity of somatostatin to inhibit basal and forskolin-stimulated adenylyl cyclase activity was decreased in vitamin A deficiency rats as compared with the control animals. All these parameters were fully restored when vitamin A was replaced in the diet. Furthermore, we found that the Gialpha1, Gialpha2 and Gialpha3 protein levels were unaltered in hippocampal membranes from rats fed a vitamin A-free diet whereas subsequent vitamin A administration to these rats caused a significant increase in the levels of Gialpha1 and Gialpha2. Altogether, the present findings suggest that dietary vitamin A levels modulate the somatostatinergic system in the rat hippocampus.

Effects of single and continuous administration of amyloid beta-peptide (25-35) on adenylyl cyclase activity and the somatostatinergic system in the rat frontal and parietal cortex.

It is unknown whether the amyloid beta-peptide (Abeta), a principal component found in extracellular neuritic plaques in the brain of patients with Alzheimer's disease (AD), is capable of altering adenylyl cyclase (AC) activity and the somatostatin (SRIF) receptor-effector system in the cerebral cortex of the patients. Therefore, the objective of this study was to investigate the effect of the beta fragment, beta (25-35), on AC activity and the somatostatinergic system in the rat frontoparietal cortex. A single dose of beta (25-35) (10microg) injected intracerebroventricularly significantly decreased the density of SRIF receptors (27.4%) and increased their affinity (32.2%) in the frontoparietal cortex. The inhibitory effect of SRIF on basal and forskolin (FK)-stimulated AC activity was significantly lower in the beta (25-35)-treated rats when compared with controls. beta (25-35) did not modify Gialpha1, Gialpha2 nor Gialpha3 levels in membranes from the frontoparietal cortex. Continuous infusion of the peptide induced a decrease in the SRIF receptor density in this brain area to a similar extent as that observed 14 days after the single administration of the peptide. Likewise, this treatment decreased the SRIF receptor density in the frontal cortex (15.3%) and parietal cortex (27.2%). This effect was accompanied by a decrease in the SRIF-mediated inhibition of FK-stimulated AC activity (from 41.6% to 25.6%) in the frontal cortex as well by a decrease in basal AC activity (from 36.9% to 31.6%) and FK-stimulated AC activity (from 35.6% to 27.1%) in the parietal cortex. Continuous infusion of Abeta (25-35) had no effect on Gialpha1, Gialpha2 or Gialpha3 levels in membranes from frontal and parietal cortex. However, this treatment caused a decrease in SRIF-like immunoreactivity content in the parietal (38.9%) and frontal (20.4%) cortex. These results suggest that Abeta might be involved in the alterations of somatostatinergic system reported in AD.

Protective effects of insulin-like growth factor-I on the somatostatinergic system in the temporal cortex of beta-amyloid-treated rats.

Insulin-like growth factor-I (IGF-I) has protective effects against beta-amyloid (Abeta)-induced neuronal cell death. Because alterations of the somatostatinergic system have been described in Alzheimer's disease, we investigated the effects of the Abeta peptide and the possible protective role of IGF-I on the somatostatinergic system of the rat temporal cortex and on cell death and phosphorylated (p)-Akt levels in this area. Abeta25-35 was administered intracerebroventricularly to male rats via an osmotic minipump over 14 days (300 pmol/day). Another group received a subcutaneous IGF-I infusion (50 microg/kg/day), concomitant with Abeta25-35 administration, whereas a third group received IGF-I alone. Abeta25-35 significantly decreased the somatostatin (SRIF)-like immunoreactive content and the SRIF receptor density, as a result of a decrease in the levels of the SRIF receptor subtype 2. The inhibitory effect of SRIF on adenylyl cyclase activity was significantly lower after Abeta25-35 infusion, whereas the levels of the inhibitory G protein subunit Gialpha1, Gialpha2 or Gialpha3 were unaltered. Cell death was increased and p-Akt levels decreased in Abeta25-35-treated animals. IGF-I administration increased immunoreactive IGF-I levels in the temporal cortex and restored all parameters affected by Abeta25-35 to baseline values. These findings suggest that IGF-I prevents the deleterious effect of Abeta25-35 on the somatostatinergic system.

Acute modulation of somatostatin receptor function by melatonin in the rat frontoparietal cortex.

Since melatonin (N-acetyl-5-methoxytryptamine) decreases locomotor activity and rearing and increases grooming behavior in a similar manner as somatostatin (SRIF), we examined if melatonin could induce these changes through somatostatinergic neurotransmission in the rat frontoparietal cortex. Male Wistar rats (200-250 g) received a single injection of melatonin (25 microg/kg per day) subcutaneously (s.c.) and were sacrificed 5 hr later. Melatonin treatment increased the number of 125I-Tyr11-SRIF receptors in frontoparietal cortical membranes without any changes in the dissociation constant (Kd). The capacity of SRIF to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity was increased in melatonin-treated rats as compared to the control animals. Melatonin administration also induced a lower AC activity, both under basal conditions and after stimulation of the enzyme via stimulatory guanine nucleotide-binding proteins (Gs), or directly with FK. Functional inhibitory guanine nucleotide-binding protein (Gi) activity was increased in frontoparietal cortical membranes from melatonin-treated rats when compared to controls. Western blot analyzes showed that melatonin administration did not alter the amount of the Gialpha1, or Gialpha3 subunits, but reduced Gialpha2 levels in frontoparietal cortical membranes. No significant changes in SRIF-like immunoreactivity content and SRIF mRNA levels were detected in this brain area after melatonin treatment. Administration of the melatonin receptor antagonist luzindole (10 mg/kg, s.c.) 30 min before melatonin injection did not change the melatonin-induced effects on the SRIF receptor effector system. In conclusion, the present results show that acute melatonin administration increases the activity of the SRIF receptor effector system and decreases Gialpha2 levels in the rat frontoparietal cortex. In addition, the coupling of Gs to AC is disturbed by melatonin.

Activation of D1 and D2 dopamine receptors increases the activity of the somatostatin receptor-effector system in the rat frontoparietal cortex.

The role of dopamine D1 and D2 receptor subtypes in the regulation, in vivo, of the somatostatin (SRIF) receptor-effector system in rat frontoparietal cortex was investigated. The D1-receptor agonist SKF 38393 (4 mg/kg) or the D2-receptor agonist bromocriptine (2 mg/kg), administered intraperitoneally to rats, increased the number of SRIF receptors without altering the affinity constant, an effect antagonized by both SCH 23390 (0.25 mg/kg) and raclopride (5 mg/kg), D1 and D2 receptor antagonists, respectively. These antagonists alone had no effect on [(125)I]Tyr(3) octreotide binding to its receptors. No change in binding was detected when the dopamine agonists were added in vitro. Basal adenylyl cyclase (AC) activity was increased by SKF 38393 treatment and decreased by bromocriptine. Octreotide (SMS 201-995)-mediated inhibition of basal and forskolin-stimulated AC was increased by SKF 38393 or bromocriptine treatment. In frontoparietal cortical slices, basal inositol-1,4, 5-triphosphate (IP(3)) levels were decreased by bromocriptine treatment but were unaffected by SKF 38393. SMS 201-995 increased the IP(3) accumulation in control, SKF 38393-, and bromocriptine-treated rats. Insofar as SRIF and dopamine appear to be involved in motor regulation and could well modulate somatosensory functions in frontal and parietal cortex, respectively, heterologous receptor regulation may have important repercussions regarding the control exerted by these neurotransmitters on frontal and parietal cortical function in the intact animal.

Diazepam attenuation of somatostatin binding and effect of somatostatin on accumulation of inositol 1,4,5-trisphosphate in the rat frontoparietal cortex.

Numerous reports in both humans and animals have confirmed that benzodiazepines produce amnesia; however, mechanisms mediating this effect are not clear. In view of the important role of brain somatostatin (SRIF) in the cognitive function of rats, this study sought to determine if the benzodiazepine, diazepam, alters somatostatinergic system in the rat frontoparietal cortex. Intraperitoneal (i.p.) administration of diazepam (5 mg/kg/day) to male Wistar rats (200-250 g) for 3 or 7 days decreased the number of SRIF receptors (26 and 37%, respectively) in synaptosomes from the frontoparietal cortex, without influencing their apparent affinity. This decrease in the tracer binding was not attributable to a direct effect of diazepam on SRIF receptors, because no decrease of SRIF binding was induced by a large concentration of diazepam (10(-4) M) when the drug was added to a preparation of synaptosomes from frontoparietal cortex of untreated rats. To determine if the effect of diazepam on SRIF binding is related to the binding of diazepam to its recognition site on the GABA(A) receptor, a benzodiazepine antagonist, 2-phenylpyrazolo[3,4-c]quinolin-3(5H)-one (CGS 8216) was administered before the diazepam injection. Pretreatment with CGS 8216 (20 mg/kg/day, i.p.) blocked completely the diazepam-induced decrease in the number of SRIF receptors. CGS 8216 alone had no observable effect. The decrease in the number of 125I-Tyr11-SRIF receptor induced by diazepam was accompanied by a decrease in the effect of SRIF, after 15 seconds of stimulation, on inositol 1,4, 5-trisphosphate (IP3) mass accumulation in the rat frontoparietal cortex at 3 (64%) or 7 days (59%) after its administration. Diazepam alone had no observable effect on mass accumulation of IP3. After 14 days of daily diazepam injections, the levels of binding of 125I-Tyr11-SRIF in the frontoparietal cortex returned to control values, coinciding with the tolerance that develops to this benzodiazepine agonists when administered chronically. The decrease in IP3 levels was still observed after 14 days (57%) diazepam administration. Diazepam and CGS 8216 did not affect SRIF-like immunoreactivity levels in the frontoparietal cortex at the three time intervals studied (3, 7 or 14 days). The alteration of frontoparietal cortex SRIF receptor-effector system after 3 or 7 days of diazepam treatment suggests that somatostatinergic neurotransmission plays a role in the mechanism of diazepam action on memory.

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning.

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.