Karin Lindström

Differences in the regional and cellular localization of c-fos messenger RNA induced by amphetamine, cocaine and caffeine in the rat

Male rats were treated i.p. with either 5 mg/kg amphetamine, 3 and 30 mg/kg cocaine or 100 mg/kg caffeine and killed after 30 min. Brains were sectioned and processed for radioactive in situ hybridization histochemistry for the labelling of either c-fos, enkephalin, substance P, neurokinin B, choline acetyltransferase, somatostatin or adenosine A2A receptor messenger RNA. The distribution of c-fos messenger RNA was investigated both at the regional level using film autoradiography, and at the cellular level using emulsion autoradiography. All drug treatments except 3 mg/kg cocaine induced an increased level of c-fos messenger RNA in cells that had a neuron-like morphology. The cells that contained the c-fos messenger RNA were identified by making pairs of 5-microns sections in which one section was processed for c-fos messenger RNA and the other was processed for one of the other messenger RNA species. After amphetamine treatment, only some 10% of the cells in the striatum were labelled, and to a variable extent. Instead there was prominent labelling of a band in the cortex that runs parallel to the cortical surface. There was also a moderate degree of labelling in the nucleus accumbens. c-fos-positive cells were substance P-positive and negative for enkephalin or A2A receptor messenger RNA. Cocaine (30 mg/kg) induced a modest labelling in the caudate-putamen, as well as in the accumbens. With cocaine treatment (30 mg/kg), about 30% of striatal neuron-like cells were c-fos labelled. Most c-fos-positive cells were substance P-positive, but none of the c-fos-positive cells were enkephalin-positive or A2A-receptor-positive. Cocaine (3 mg/kg) had no significant effect on c-fos. Caffeine gave rise to a strong hybridization signal in the caudate-putamen, particularly the dorsolateral part. No other region examined differed significantly from control. With caffeine treatment, about 73% of neuron-like cells were c-fos labelled in the lateral striatum, but labelling was much less pronounced in the medial part or in the accumbens. c-fos-labelled cells were found in enkephalin-positive and enkephalin-negative, substance P-positive and substance P-negative, neurokinin B-positive and neurokinin B-negative groups. No choline acetyltransferase-positive or somatostatin-positive cells were found that were also c-fos-positive with any of the treatments. We conclude that each of the different CNS stimulant drugs induces a highly specific pattern of c-fos messenger RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Receptors Mediating Cyclic AMP Productioin the Rat Hippocampus

Abstract: In the transversely cut rat hippocampus, adenosine caused a dose‐dependent increase in the accumulation of [3H]cyclic AMP from [3H]ATP. Adenosine breakdown products were inactive. AMP was somewhat less effective than adenosine, and its effect could be partially, but not completely, abolished by α,β‐methylene‐ADP and GMP, which inhibited its metabolism by 5′‐nucleotidase. The effect of adenosine was unaffected by inhibitors of adenosine deaminase, but enhanced by several inhibitors of adenosine uptake. Some analogues of adenosine, including N6‐phenylisopropyladenosine (PIA), 2‐chloroadenosine and adenosine 5′‐ethylcarboxamide (NECA), were more active than adenosine, whereas others such as 2‐deoxyadenosine and 9‐(tetrahydro‐2‐furyl)adenine (SQ 22536) actually inhibited the response. The effect of PIA was highly stereospecific. The action of adenosine was inhibited by several alkylxanthines, the most potent of which was 8‐phenyltheophylline. [3H]Cyclohexyladenosine (CHA) bound specifically to cell membranes from the rat hippocampus. The extent of binding was similar to that found in other cortical areas. The relative potency of some adenosine analogues and alkylxanthines to displace labelled CHA was essentially similar to their potency as effectors of the cyclic AMP system. Adenosine contributed to the cyclic AMP‐elevating effect of α‐adrenoceptor‐stimulating drugs and several amino acids, but not to that seen with isoprenaline. The cyclic AMP increase seen following depolarization was only partially adenosine‐dependent. The present results demonstrate that the rat hippocampus contains adenosine receptors mediating cyclic AMP accumulation and that these receptors have similar characteristics to those mediating pyramidal cell depression. Adenosine‐induced cyclic AMP accumulation may be used as a biochemical correlate to electrophysiology and as a convenient parameter to assess the influence of drugs on adenosine mechanisms in the rat hippocampus.

On the mechanism by which methylxanthines enhance apomorphine-induced rotation behaviour in the rat

Methylxanthines, such as caffeine and theophylline, potentiate the rotation behaviour induced by dopamine receptor agonists in rats with unilateral lesions of the nigro-striatal pathway. In the present study we have examined the possibility that interaction with central adenosine mechanisms could influence rotation behaviour. Under in vitro conditions adenosine and N6-phenylisopropyl-adenosine (PIA) stimulate cyclic AMP accumulation. This effect was enhanced by the phosphodiesterase inhibitor rolipram, but blocked by alkylxanthines such as caffeine, theophylline and, particularly, 8-phenyl-theophylline. Rotation behaviour induced by apomorphine (0.05 mg/kg), was inhibited by PIA and rolipram and by a low dose of the adenosine deaminase inhibitor EHNA (2 mg/kg). By contrast, theophylline and 8-phenyl-theophylline caused a potentiation. The former drug stimulated rotation behaviour per se, while the latter did not. 8-Phenyl-theophylline entered the brain poorly and its concentration in brain it was less than 1/10 of theophylline. It is concluded that theophylline does not potentiate rotation behaviour secondarily to inhibition of phosphodiesterase. Antagonism of endogenous adenosine may partly explain the effect of methylxanthines. Possibly, some as yet unknown mechanism may also contribute to the effects of xanthine-derivatives on rotation behaviour.

A1 and A2A adenosine receptors and A1 mRNA in mouse brain: effect of long-term caffeine treatment

The effect of oral treatment with caffeine, in doses that are known to produce marked adaptive effects, was investigated on A1 and A2A receptors in the mouse brain. Caffeine (0.1, 0.3 or 1 g/l) was added to the drinking water and the animals were sacrificed after a 14-day treatment period. Ligand binding to A1 receptors was studied, using quantitative autoradiography, with the agonist [3H]cyclohexyladenosine (CHA) and the antagonist [3H]1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Caffeine did not remain in the sections during the autoradiography experiments. Caffeine treatment (1 g/l, but not 0.1 or 0.3 g/l) tended to increase [3H]CHA binding to the CA3 subfield of the hippocampus, but in no other region studied. There was no change in the number of A1 receptors since [3H]DPCPX binding to the CA3, cerebral and cerebellar cortex was not influenced by caffeine treatment. There was similarly no change in the ability of CHA to displace [3H]DPCPX binding, suggesting that there are no major changes in the proportion of A1 receptors that are coupled to G-proteins. mRNA for the A1 receptor, measured by in situ hybridization, did not differ significantly between caffeine-treated and control mice in the structures examined. Thus, higher doses of caffeine can cause an increase in A1 agonist binding without a corresponding change in A1 mRNA or in A1 antagonist binding, suggesting that the adaptive changes seen upon prolonged caffeine treatment may be in sites different from A1 receptors. Caffeine (1 g/l) increased A2A receptors in the striatum measured as binding of the agonist [3H]CGS 21680 suggesting that up-regulation of A2A receptors may be an adaptive effect of caffeine intake.

Propentofylline and Other Adenosine Transport Inhibitors Increase the Efflux of Adenosine Following Electrical or Metabolic Stimulation of Rat Hippocampal Slices

Abstract: Propentofylline is a novel neuroprotective agent that has been shown to act as an adenosine transport inhibitor as well as an adenosine receptor antagonist. In the present series of experiments we have compared the effects of propentofylline with those of known adenosine transport inhibitors and receptor antagonists on the formation of adenosine in rat hippocampal slices. The ATP stores were labeled by incubating the slices with [3H]‐adenine. The total 3H overflow and the overflow of endogenous and 3H‐labeled adenosine, inosine, and hypoxanthine were measured. Adenosine release, secondary to ATP breakdown, was induced both by hypoxia/hypoglycemia and by electrical field stimulation. Propentofylline (20–500 µM) increased the release of endogenous and radiolabeled adenosine, without increasing the total release of purines. Thus, the drug altered the pattern of released purines, i.e., increasing adenosine and decreasing inosine and hypoxanthine. This pattern, which was observed when purine release was induced both by electrical field stimulation and by hypoxia/hypoglycemia, was shared by the nucleoside transport inhibitor dipyridamole (1 µM) and by mioflazine (1 µM) and nitrobenzylthioinosine (1 µM). By contrast, other xanthines, including theophylline (100 µM) and 8‐cyclopentyltheophylline (10 µM), enprofylline (100 µM), or torbafylline (300 µM), if anything, increased the total release of purines without alterations of the pattern of release. These results indicate that nucleoside transport inhibitors can decrease the release of purines from cells and at the same time increase the concentration of extracellular adenosine, possibly by preventing its uptake and subsequent metabolism. This change in purine metabolism may be beneficial with regard to cell damage after ischemia. The results also indicate that propentofylline behaves in such a potentially beneficial manner.

The selective adenosine A2A receptor antagonist SCH 58261 discriminates between two different binding sites for [3H]-CGS 21680 in the rat brain

We have used quantitative autoradiography to further examine two previously described binding sites for [3H]-CGS 21680 in cortical regions and in striatum, respectively. The striatal binding sites largely represent classical adenosine A2A receptors whereas the cortical sites show characteristics that differ from those of recognised adenosine receptors. A recently developed non-xanthine A2A receptor antagonist SCH 58261 displaced the binding of [3H]-CGS 21680 from the A2A receptors in striatum with an estimated Ki value of 2.4 nM, but was more than 1000-fold less potent in displacing its binding from cortex. Conversely, the adenosine analogue 2-chloro-NECA was found to be some 10 times more potent in displacing CGS 21680 from the cortical binding sites than from A2A receptors. The results provide additional evidence that CGS 21680 binds not only to classical A2A receptors, but also to sites that differ from defined adenosine receptors. They also suggest that effects of CGS 21680 observed in the presence of SCH 58261 might reveal the functional significance (if any) of these sites.

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6‐[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800‐fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 µM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine‐rich regions of the rat brain, with a KD value of 1.4 (0.8–1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H]SCH 58261 with the following estimated Ki values (nM): 2‐hex‐1‐ynyl‐5′‐N‐ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N6‐cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µM) or Mg2+ (10 mM). The present results show that in its tritium‐labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.

Lack of tolerance to motor stimulant effects of a selective adenosine A2A receptor antagonist

It is well known that tolerance develops to the actions of caffeine, which acts as an antagonist on adenosine A(1) and A(2A) receptors. Since selective adenosine A(2A) antagonists have been proposed as adjuncts to 3,4-dihydroxyphenylalanine (L-DOPA) therapy in Parkinsons disease we wanted to examine if tolerance also develops to the selective A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo [1,5-c]pyrimidine (SCH 58261). SCH 58261 (0.1 and 7.5 mg/kg) increased basal locomotion and the motor stimulation afforded by apomorphine. Neither effect was subject to tolerance following long-term treatment with the same doses given intraperitoneally twice daily. There were no adaptive changes in A(1) and A(2A) adenosine receptors or their corresponding messenger RNA or in dopamine D(1) or D(2) receptors. These results demonstrate that the tolerance that develops to caffeine is not secondary to its inhibition of adenosine A(2A) receptors. The results also offer hope that long-term treatment with an adenosine A(2A) receptor antagonist may be possible in man.

New treatment for IgA nephropathy: enteric budesonide targeted to the ileocecal region ameliorates proteinuria

BACKGROUND Systemic corticosteroid treatment has been shown to exert some protection against renal deterioration in IgA nephropathy (IgAN) but is not commonly recommended for long-term use due to the well-known systemic side effects. In this study, we investigated the efficacy and safety of a new enteric formulation of the locally acting glucocorticoid budesonide (Nefecon®), designed to release the active compound in the ileocecal region. The primary objective was to evaluate the efficacy of targeted release budesonide on albuminuria. METHODS Budesonide 8 mg/day was given to 16 patients with IgAN for 6 months, followed by a 3-month follow-up period. The efficacy was measured as change in 24-h urine albumin excretion, serum creatinine and estimated glomerular filtration rate (eGFR). RESULTS The median relative reduction in urinary albumin excretion was 23% during the treatment period (interquartile range: -0.36 to -0.04, P = 0.04) with pretreatment values ranging from 0.3 to 6 g/24 h (median: 1.5 g/24 h). The median reduction in urine albumin peaked at 40% (interquartile range: -0.58 to -0.15) 2 months after treatment discontinuation. Serum creatinine was reduced by 6% (interquartile range: -0.12 to -0.02; P = 0.003), and eGFR [Modification of Diet in Renal Disease (MDRD)] increased ∼8% (interquartile range: 0.02-0.16, P = 0.003) during treatment. No major corticosteroid-related side effects were observed. CONCLUSIONS In the present pilot study, enteric budesonide targeted to the ileocecal region had a significant effect on urine albumin excretion, accompanied by a minor reduction of serum creatinine and a modest increase of eGFR calculated by the MDRD equation, while eGFR calculated from Cockcroft-Gault equation and cystatin C was not changed. Enteric budesonide may represent a new treatment of IgAN warranting further investigation.

Effect of adenosine receptor agonists and other compounds on cyclic AMP accumulation in forskolin-treated hippocampal slices

Summary1.The effect of adenosine analogues and some putative neurotransmitters have been studied on cyclic AMP accumulation in rat hippocampal slices treated with the adenylate cyclase activator forskolin.2.The effects of PGE2 and histamine were potentiated by forskolin (0.1 μM). Isoprenaline and NECA had essentially additive effects with 0.1 μM forskolin and serotonin (above 10−4 M) inhibited forskolin-stimulated cyclic AMP accumulation.3.The A1-adenosine receptor selective adenosine analogue R-PIA inhibited forskolin stimulated cyclic AMP accumulation in low doses and stimulated in high. NECA, adenosine and 2-chloroadenosine uniformly stimulated cyclic AMP accumulation. 2′,5′-dideoxyadenosine inhibited, but only at high concentrations.4.Both the stimulatory and the inhibitory effects of R-PIA were antagonized by 8-phenyltheophylline (10 μM). Enprofylline (100 μM) selectively inhibited the stimulatory effect. In the presence of enprofylline both 2-chloroadenosine showed an inhibitory effect on cyclic AMP accumulation.5.It is concluded that the forskolin-treated rat hippocampal slice is a useful preparation to study both stimulatory and inhibitory effects of transmitters and modulators on adenylate cyclase. The results also show that the rat hippocampus has both A1-receptors that are linked to inhibition of cyclic AMP accumulation and A2-receptors that are linked to stimulation. Furthermore, enprofylline is shown to selectively antagonize the stimulatory response, revealing inhibitory effects of compounds such as 2-chloroadenosine and adenosine.