The anxiolytics CI-988 and chlordiazepoxide fail to reduce immediate early gene mRNA stimulation following exposure to the rat elevated X-maze.

This study uses immediate early transcription factor gene expression to map neuronal activation after a single exposure to the elevated X-maze. Exposure to this novel environment leads to widespread upregulation in the gene expression of c-fos, NGFI-A and NGFI-B (the nerve growth factor induced genes), but not c-jun nor jun B as shown by in situ hybridization and northern blot analysis. Changes in c-fos were evident after just 5 min exposure to the maze. The cholecystokininB receptor antagonist, CI-988, given intraperitoneally at 1 mg/kg 40 min prior to exposure to the X-maze demonstrated an anxiolytic profile without affecting overall movement around the maze, however it did not reduce the increased levels of gene expression with the methodology used. Likewise the anxiolytic benzodiazepine, chlordiazepoxide at 3 mg/kg did not reduce gene expression. It is concluded that a reduction in an index of behavioural stress/anxiety produced by anxiolytic agents is not concomitantly followed by a detectable reduction in immediate early gene induction.

Expression of immediate early genes in rat gastric myenteric neurones: a physiological response to feeding.

1. Expression of the immediate early genes c-fos, c-jun and c-myc in rat stomach in response to feeding and gastric distension was examined by Northern blot analysis and in situ hybridization. 2. Refeeding of fasted rats induced a transient increase in c-fos mRNA abundance in gastric corpus and antrum that was sixfold within 15 min and declined within 4 h. The response was not mediated by gastrinergic or muscarinic cholinergic mechanisms; it was reduced but not abolished by hexamethonium. No changes in expression of c-jun, c-myc or the constitutively expressed protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were observed. 3. In conscious rats prepared with a gastric fistula, gastric distension with nutritive and non-nutritive solutions at a physiological pressure for 30 min induced expression of c-fos, c-jun and c-myc, but not GAPDH. 4. Messenger RNA encoding c-fos was localized by in situ hybridization to gastric myenteric neurones of animals that underwent gastric distension, but not of undistended controls. 5. The results suggest that expression of c-fos in gastric myenteric neurones is an early response to the physiological stretching of the stomach wall that accompanies feeding. With supraphysiological distension, other immediate early genes may be recruited.

Changes in hypothalamic cholecystokininA and cholecystokininB receptor subtypes and associated neuropeptide expression in response to salt-stress in the rat and mouse.

This study demonstrates cholecystokinin receptor plasticity in response to salt-loading in the rat and mouse hypothalamus. It identifies, for the first time, the cholecystokinin receptor subtypes involved, firstly by receptor autoradiography and secondly by in situ hybridization. Both species showed increases in hypothalamic [125I]Bolton Hunter-cholecystokinin-8 binding. Co-incubation with the specific cholecystokininA and cholecystokininB antagonists, devazepide and CI-988, indicated that in the rat cholecystokininB receptor binding markedly increased, with a small increase in cholecystokininA receptor binding. In the mouse the response was comprised solely of cholecystokininA receptors. In situ hybridization studies were carried out on a range of peptide messenger ribonucleic acids after salt-loading. In the rat large increases in hypothalamic gene expression were detected for oxytocin, vasopressin, corticotrophin-releasing factor and preprocholecystokinin. In the mouse only vasopressin messenger ribonucleic acid increased, whilst hypothalamic oxytocin, preprocholecystokinin and corticotropin-releasing factor remained unchanged. However, corticotrophin-releasing factor messenger ribonucleic acid increased in the mouse amygdala. In situ hybridization was performed using oligonucleotide probes specific for either the cholecystokininA or cholecystokininB receptor messenger ribonucleic acid, and this showed good agreement with the receptor autoradiography. CholecystokininB receptor expression was upregulated in the rat hypothalamus along with a small but significant increase in cholecystokininA receptors. In the mouse only cholecystokininA receptor expression was increased. In addition to these molecular changes rats lost about 25% of their body weight during six days of salt-challenge, whilst mice continued to grow in line with controls. This work demonstrates differential changes in cholecystokinin receptor subtype binding between the rat and the mouse. It represents the first report of differential changes in cholecystokininA and cholecystokininB receptor messenger ribonucleic acids within the brain, and shows that cholecystokinin receptors within the rodent hypothalamus are capable of plastic responses to chronic osmotic stress.

c-fos antisense oligodeoxynucleotide increases formalin-induced nociception and regulates preprodynorphin expression.

Rats, receiving an intrathecal pretreatment of oligodeoxynucleotide complementary to c-fos mRNA (antisense), showed no increases in Fos protein or preprodynorphin messenger RNA in the outer laminae of the lumbar spinal cord when challenged 4 h later with a 50 microliters intraplantar injection of 5% formalin. Animals pretreated with saline or sense oligodeoxynucleotide showed marked increases in Fos protein (2 h after formalin challenge) and preprodynorphin mRNA (20 h after formalin challenge) in the lumbar region of the cord ipsilateral to the side of the injection. The behavioural consequences of antisense pretreatment were an increase in the formalin-induced licking/biting responses during the tonic, but not the acute phase. These observations could be interpreted as representing a sequence of events beginning with the formalin-induced increase in the transcription factor Fos, which in turn increases the synthesis of preprodynorphin messenger RNA resulting in the production of the dynorphin opioid peptides which then exert a modulatory antinociceptive action.

Evidence that cholecystokinin induces immediate early gene expression in the brainstem, hypothalamus and amygdala of the rat by a CCKA receptor mechanism.

The effect of sulphated cholecystokinin octapeptide (CCK-8S) on immediate early gene expression in the rat CNS was investigated using the technique of in situ hybridization. A rapid and transient induction of c-fos, NGFI-A and NGFI-B (nerve growth factor-induced genes A and B) mRNA was demonstrated in the nucleus tractus solitarius (NTS), area postrema (AP), hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and central nucleus of the amygdala, following peripheral administration of CCK-8S (1-100 micrograms/kg i.p.). In contrast, levels of c-jun, junB and junD mRNA were unaffected by the peptide. The closely related decapeptide, caerulein (50 micrograms/kg i.p.), induced the same pattern of IEG expression as CCK-8S, whereas the desulphated octapeptide, CCK-8DS (50 micrograms/kg i.p.), had no effect on levels of mRNA for any IEG studied. Expression of IEG mRNA in these areas was suppressed by bilateral subdiaphragmatic vagotomy, and by pretreatment with the selective CCKA receptor antagonist, devazepide (0.1 and 1 mg/kg i.p.). In contrast, CCK-8S induction of IEG mRNA was not blocked by pretreatment with the selective CCKB receptor antagonist, CI-988 (1 or 10 mg/kg i.p.). In addition, the selective CCKB receptor agonists, CCK-4 (50 micrograms/kg i.p.) or pentagastrin (2 mg/kg i.p.), failed to induce IEG expression in any of the areas studied. These results suggest that systemic CCK-8S primarily acts via CCKA receptors on vagal afferents to stimulate IEG mRNA expression in the rat CNS.

The effect of dizocilpine and enadoline on immediate early gene expression in the gerbil global ischaemia model.

Increases in mRNA levels for c-fos, c-jun, junB, hsp70 and NGFI-A were observed in the dentate gyrus of the hippocampus following 7 min ischaemia in the Mongolian gerbil. The response was rapid and transient (30 min to 4 hr) for NGFI-A, junB and c-fos mRNA. In contrast c-jun mRNA remained increased for several hours. Hsp70 increased in the dentate gyrus 1 hr after the insult, returned to control values at 4 hr and showed a secondary increase at 24 hr. At 24 hr increased hsp70 mRNA was observed in other regions of the CNS, i.e. CA1, CA2, CA3 and cortex. The non-competitive NMDA receptor antagonist, dizocilpine, attenuated the increases in IEG expression and was neuroprotective. In contrast the kappa opiate receptor agonist, enadoline, protected the CA1 neurones from degeneration but did not inhibit the increased levels of IEG mRNA. Increases in hsp70 mRNA were reduced to baseline by both enadoline and dizocilpine. These results suggest that inhibition of IEG expression is not a prerequisite for neuroprotection. However, hsp70 was predictive of neuronal protection and may be a useful assay in this and related models.

Solubilization of the N-methyl-D-aspartate receptor channel complex from rat and porcine brain.

The N-methyl-D-aspartate (NMDA) receptor complex as defined by the binding of [3H]MK-801 has been solubilized from membranes prepared from both rat and porcine brain using the anionic detergent deoxycholate (DOC). Of the detergents tested DOC extracted the most receptors (21% for rat, 34% for pig), and the soluble complex, stabilized by the presence of MK-801, could be stored for up to 1 week at 4 degrees C with less than 25% loss in activity. Receptor preparations from both species exhibited [3H]MK-801 binding properties in solution very similar to those observed in membranes (Bmax = 485 +/- 67 fmol/mg of protein, KD = 11.5 +/- 2.9 nM in rat; Bmax = 728 +/- 108 fmol/mg of protein, KD = 7.1 +/- 1.6 nM in pig, n = 3). The pharmacological profile of the solubilized [3H]MK-801 binding site was virtually identical to that observed in membranes. The rank order of potency of: MK-801 greater than (-)-MK-801 = thienylcyclohexylpiperidine greater than dexoxadrol greater than SKF 10,047 greater than ketamine, for inhibition of [3H]MK-801 binding, was observed in all preparations. The receptor complex in solution exhibited many of the characteristic modulations observed in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The possible involvement of changes in phosphoinositol turnover in the responses of renal sodium transport to noradrenaline.

Noradrenaline stimulated the formation of [3H]inositol monophosphates from 3H-inositol-phospholipids in rat kidney cortex slices. This response was inhibited by the alpha 1-adrenoceptor antagonist prazosin but not by the alpha 2-adrenoceptor antagonist yohimbine, suggesting that the response was mediated via an alpha 1-adrenoceptor. This is in keeping with the ability of noradrenaline to increase sodium transport in this tissue by alpha 1-adrenoceptor stimulation.

Differences between high-affinity forskolin binding sites in dopamine-rich and other regions of rat brain.

[3H]Forskolin bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into one rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebellum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.

Synergistic interactions between forskolin, isoprenaline and substance P as secretagogues in rat parotid glands.

Substance P, forskolin and isoprenaline stimulated rat parotid alpha-amylase secretion in vitro. Synergistic responses were observed with combinations of any two of the three secretagogues such that subthreshold doses of one caused a pronounced shift in the dose-response curve to the second. This potentiation of secretion could neither be explained by an interaction at the receptor recognition binding site, as identified by ligand binding, nor wholly by interactions in second messenger systems. Thus forskolin and isoprenaline were unable to alter substance P-induced changes in phosphatidylinositol metabolism. Similarly substance P was without effect on forskolin or isoprenaline-stimulated cAMP production. In contrast the potentiation of isoprenaline-induced amylase secretion by forskolin was preceded by a marked enhancement of cAMP production suggesting that the activation of the adenylate cyclase complex is reflected in the cellular response.

Identification of selective, high affinity [125I]-angiotensin and [125I]-bradykinin binding sites in rat intestinal epithelia.

Specific [125I]-angiotensin II (AII) and [125I]-bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon. These high affinity intestinal sites exhibited KD values of 0.64 +/- 0.16 nM for [125I]-AII and 0.69 +/- 0.13 nM for [125I]-Bk, which were similar to those for [125I]-AII (0.85 nM) and [125I]-Bk binding sites (1.03 nM) previously identified in renal cortex epithelia. Specific [125I]-AII binding capacity was only 19.77 +/- 2.74 fmol mg-1 in small intestine and 11.31 +/- 2.66 fmol mg-1 in descending colon epithelia while a larger population, 332.0 +/- 72.9 fmol-mg-1 of specific [125I]-Bk sites were identified in epithelial membranes from small intestine. Significant hydrolysis of both free [125I]-AII and [125I]-Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of KD and Bmax quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the KD value for both peptides. Loss of membrane bound peptide, particularly of [125I]-AII, may indicate that the calculated Bmax value is an underestimation. Despite the rapid degradation of unbound [125I]-AII and [125I]-Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [125I]-AII binding was [Sar1, Leu8]-AII greater than or equal to [Sar1, Thr8]-AII greater than or equal to AII greater than [Sar1, Ile8]-AII greater than or equal to [Des, Asp1, Ile8] AII greater than AIII. Specific [125I]-Bk binding was also highly selective, the order of potency being Phe8-Bk greater than or equal to Tyr8-Bk greater than or equal to Lys-Bk much greater than Des, Arg1-Bk. AII exhibited an IC50 of greater than 1mM for specific [125I]-Bk binding and likewise Phe8-Bk for specific [125I]-AII binding.

The characterization of [3H]sulpiride binding sites in pig striatal membranes.

Pig striatal membranes have [3H]sulpiride-binding sites similar to those identified in rat striatal membranes. The pharmacological profile indicates that this binding is to dopamine receptors. Agonist displacement of [3H]sulpiride binding in pig striatal membranes is subject to guanine nucleotide regulation. This effect is mimicked by heat treatment. N-ethyl maleamide (20 microM) and dithioerythritol (3 mM) decrease agonist affinity for the [3H]sulpiride-binding site in pig striatal membranes without significantly affecting maximal displacement.

The effect of cholecystokinin octapeptide on rat striatal 3H spiperone binding.

In the rat striatum sulphated CCK8 has no significant effect on equilibrium binding of 3H-spiperone but has a considerable, although transient, effect under non-equilibrium conditions. Under non-equilibrium conditions (during the association phase of ligand binding) and at high ligand concentrations (1 nM), CCK8 displaces specific binding and at low ligand concentrations (0.1 nM) CCK8 enhances specific binding. CCK8 has no effect on 3H-spiperone dissociation kinetics.

Inactivation of [125I]-angiotensin II binding sites in rat renal cortex epithelial membranes by dithiothreitol.

Preincubation of renal epithelial membranes with DTT produced a dose-dependent inhibition of specific [125I]-angiotensin binding, with an IC50 of 1 mM and total loss of binding at 5-10 mM DTT. Inactivation of specific [125I]-angiotensin II binding by DTT was temperature sensitive; the t1/2 at 22 degrees was 6 min compared with 30 min at 4 degrees. A rapid inactivation rate was dependent on the presence of NaCl. In the presence of 120 mM NaCl the t1/2 for inactivation by DTT was 6 min and 33 min in the absence of NaCl. Protection against DTT inactivation was obtained by preincubating membranes with unlabelled angiotensin II greater than angiotensin I greater than renin substrate while the dipeptide, Ileu-His was only effective in protecting the binding site at high concentrations (10 mM). Preincubations with DTT (1 mM) caused a 43% decrease in Bmax from 217.0 +/- 39.5 to 123.7 +/- 30.9 fmol bound/mg protein while the KD was not significantly affected.

Studies of the mechanism of noradrenaline stimulation of fluid absorption by rat jejunum in vitro.

The everted-sac technique was used to study the mechanism of action of noradrenaline on fluid absorption by rat jejunum. Noradrenaline (10(-3) M) significantly stimulated fluid absorption and this effect was dependent on the presence of calcium ions in the serosal fluid. Strontium, but not magnesium could substitute for calcium. Verapamil, manganese and neodymium, all inhibitors of calcium transport, blocked noradrenaline-stimulated fluid absorption when present in the serosal compartment without any effect on basal or glucose-stimulated absorption. Inhibitors of the translation stage of protein synthesis inhibited the response whereas blocking the transcription stage of protein synthesis was without effect. The noradrenaline response was not attenuated by tetrodotoxin suggesting that the response is not indirect due to noradrenaline altering endogenous intestinal nervous activity. It is concluded that noradrenaline acts by opening calcium channels in the basolateral membranes of epithelial cells, resulting in an influx of calcium which stimulates ribosomal protein synthesis to produce proteins involved in fluid transport.

Location of [125I]-angiotensin II receptors on rat kidney cortex epithelial cells.

Angiotensin II (AII) stimulates active Na+ extrusion from Na+ loaded renal cortex slices. Specific high affinity [125I]-AII binding sites in partially purified basolateral and brush-border epithelial membranes exhibit a KD of 0.88 nM and Bmax of 321.13 fmol mg-1 protein. Separation and purification of brush-border membranes yielded high affinity [125I]-AII binding sites with KD of 1.02 nM and Bmax of 56.6 fmol mg-1 protein. Angiotensin II receptors of the same affinity are present on renal cortex brush-border and basolateral membranes but a greater proportion are located on the latter. These receptors may be involved in the direct control of Na+ and water transport by AII.

The binding of [3H]prazosin and [3H]clonidine to rat jejunal epithelial cell membranes.

The binding of [3H]prazosin and [3H]clonidine to rat jejunal epithelial cell membranes has been studied. The membrane preparation was enriched in baso-lateral components as determined by Na+, K+ ATPase and alkaline phosphatase activities. The membranes possessed two saturable specific binding sites for [3H]prazosin, a high affinity (Kd 0.17 nM) low capacity (Bmax 27.3 fmole bound per mg protein) and a low affinity (Kd 5.0 nM) high capacity (Bmax 276 fmole bound per mg protein) site. The specificity of both sites was similar and was related to alpha 1-adrenoceptors. [3H]Clonidine bound to the membranes in a saturable fashion (Kd 7.3 nM). The specificity of this site was related to alpha 2-adrenoceptors. The [3H]clonidine binding site was present in the membranes in much lower density (Bmax 22.8 fmole bound per mg protein) suggesting that alpha 1-adrenoceptors predominate in this tissue.