Nucleus locus ceruleus: new evidence of anatomical and physiological specificity

The role of adenosine and its nucleotides in central synaptic transmission.

Publisher Summary This chapter discusses the physiological role of adenosine in the central nervous system (CNS). In particular, the chapter's attention is directed to the actions of adenosine and related purines in the CNS. One of the major advances in the understanding of purine actions in the central nervous system is the characterization of high affinity specific receptors for purines in brain membranes. The physiological actions of purines, the receptors and biochemical actions that underlie these functional responses and behavioral responses to purinergic drugs are considered. There are three different adenosine receptor sites in brain, which can be distinguished primarily by their effects upon adenylate cyclase, their pharmacological properties, and by their ability to bind labeled analogs of adenosine. One of the best-characterized physiological actions of adenosine is the inhibition of the release of neurotransmitters, an action that has been unequivocally established at many peripheral synapses. If one thing remains clear, it is that purines have a multitude of complex actions at every level, including the biochemical, physiological, and behavioral. There remains little question, but that adenosine or other purines constitute important and rather ubiquitous regulators of neuronalactivity in brain. Despite the evidence that purines play a significant role in neural function, it has remained difficult to define the functional role(s) of purines in brain.

The physiological role of adenosine in the central nervous system.

Adenosine receptors in the central nervous system: relationship to the central actions of methylxanthines

Purinergic signalling: pathophysiological roles.

1. 1. Adrenergic agonists (adrenaline, noradrenaline, isoproterenol and phenylephrine) stimulate the activity of (Na + K + ) ATPase (EC 3.6.1.3) in rat cerebral cortical synaptosomal fractions. 2. 2. The potencies of the agonists on the stimulation of the enzyme arranged in descending order are adrenaline ⩾ noradrenaline > isoproterenol > phenylephrine. 3. 3. The response to noradrenaline can be antagonized by both α- and β-adrenergic blockers. 4. 4. We conclude that the stimulation of cerebral (Na + K + ) ATPase by biogenic amines could be a receptor-mediated process. 5. 5. The results also indicate that it is possible that both α- and β-receptors activate a common mechanism.

Effects of α- and β-adrenergic blocking agents on the biogenic amine stimulated (Na+K+) ATPase of rat cerebral cortical synaptosomal membrane

1. 1. Thirteen benzodiazepines have been tested for their ability to inhibit the uptake of adenosine by rat brain synaptosomes. 2. 2. All of these benzodiazepines were able to inhibit adenosine uptake with IC 20 values ranging from 5 × 10 −9 M (clonazepam) to 2.0 × 10 −5 M (RO 11-6893). 3. 3. Clonazepam, nitrazepam, lorazepam, RO 11-6896, diazepam, flunitrazepam, medazepam, and flurazepam were the most potent compounds, having IC 20 values of less than 10 −6 M. Adenosine uptake would therefore be appreciably reduced at the levels achieved with therapeutic doses (circa 1 μM for diazepam). 4. 4. The d-steroisomer RO 11-6896 was approximately 200 times more active than the 1-isomer, RO 11-6893. 5. 5. The high IC 50 values (10 −5 −10 −3 M) indicate that dose response curves for the benzodiazepines are rather shallow. 6. 6. The results are consistent with the hypothesis that benzodiazepines exert some of their actions via an enhancement of the extracellular levels of adenosine.

Inhibition of adenosine uptake into rat brain synaptosomes by the benzodiazepines.

1. 1. The stimulation by biogenic amines of (Na+-K+) ATPase in a rat brain cortical homogenate has been examined. 2. 2. It was found that amine stimulation of (Na+-K+) ATPase activity possibly involves a “catecholic receptor”. 3. 3. The binding affinity of the amines to the “receptor” as measured by the concentration of amine required to produce a 50% stimulation of the enzyme was; noradrenaline (3.1 × 10−7 M), adrenaline (3.1 × 10−6 M) and isoprenaline (6.7 × 10−6 M). Phenylephrine was ineffective. 4. 4. The α- and β-adrenergic blockers, phentolamine and propranolol stimulated enzyme activity at 10−5 and 10−6 M, however, they both inhibited (Na+-K+) ATPase activity at a 10−3M concentration. 5. 5. Both phentolamine and propranolol (10−5M) abolished the enzyme stimulation by 10−6M noradrenaline. This blocking effect of the antagonists could be reversed by increasing the noradrenaline concentration to 10−5M suggesting that noradrenaline may have a higher binding affinity to the “catecholic receptor” than do the adrenergic antagonists. 6. 6. Isoprenaline (10−5M) did not reverse the blocking effect of either phentolamine or propranolol (10−5M). However, increasing the isoprenaline concentration to 10−4M reversed the effect of the blockers, suggesting that isoprenaline may have a lower affinity to the “receptor” than noradrenaline. 7. 7. In conclusion, it appears that noradrenaline stimulation of (Na+-K+) ATPase in rat brain homogenate may be mediated by a “catecholic receptor” whose activation can be antagonized by the α-and β-blockers, phentolamine and propranolol.

Receptor-mediated noradrenaline stimulation of (Na+-K+) ATPase in rat brain cortical homogenates

The suggestion has beenmade in earlier publications that benzodiazepines may exert some of their therapeutic effects by potentiating the pharmacological actions of endogenously released adenosine (Phillis 1979; Phillis et al 1979a). Adenosine and the adenine nucleotides are highly potent depressants of the firing of many central neurons. Diazepam potentiates the depressant actions of adenosine and 5’-adenosine monophosphate (5’AMP) on the spontaneous firing of cerebral cortical neurons and in largeramounts it evokes a similar depression of neuronal firing. The depressant actions of another benzodiazepine, flurazepam, are inhibited by the adenosine antagonist, theophylline. Both sets of observations are consistent with the postulate that benzodiazepines exert their actions on cortical neurons through a purinergic link. Mah & Daly (1976) have shown that diazepam inhibits the uptake of adenosine by rat brain slices but in their experiments high concentrations (5 x 10-5-2 x lo-‘ M) of diazepam were required to elicit such an inhibition. These concentrations are well above those which are likely to occur in the brain or in plasma (up to 1 0 6 ~ ) during the therapeutic administration of diazepam to man (Hillestad et al 1974; Skolnick et a1 1979). To validate our hypothesis that diazepam may elicit at least some of its therapeutic actions by inhibiting adenosine uptake, it is necessary to demonstrate that this agent, in therapeutically relevant concentrations, can in fact significantly affect such uptake. The results presented in this communication show that diazepam, at concentrations as low as 10-8-10-7 M, reduces adenosine uptake. A second benzodiazepine, flurazepam, which is less potent than diazepam as a therapeutic agent (Mohler & Okada 1978), also inhibited uptake, but somewhat less effectively. Synaptosomes were prepared by the method of Gray & Whittaker (1962) as modified by White (1975). Briefly, 4 male Wistar rats were killed by cervical dislocation. The cerebral cortices were removed, weighed and homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose solution, p H 7.5. The homogenate was centrifuged at 1OOO g for 10 min. The debris was discarded and the remaining supernatant was centrifuged again a t 12 OOO g for 20 min at 4°C. The resultant pellet was suspended in 12 ml of ice-cold 0.32 M sucrose solution, p H 7.5, and transferred to a discontinuous sucrose gradient and centrifuged at 150 OOO g for 60 min. The synaptosomal fraction was collected and diluted to 40 ml with 0.32 M sucrose solution and then centrifuged at 20 000 g for 20 min at 4°C. The synaptosomal pellet

Diazepam and flurazepam inhibit adenosine uptake by rat brain synaptosomes.

1. 1. The proposal is developed that endogenously released adenosine may regulate the vascular supply to neoplastic tissue. Evidence supporting this suggestion includes findings that agents which potentiate adenosine's action enhance tumor growth and the adenosine antagonists reduce the size of primary tumours and the size and number of metastases.

P.H. Wu

Papers

Effects of α- and β-adrenergic blocking agents on the biogenic amine stimulated (Na+K+) ATPase of rat cerebral cortical synaptosomal membrane

Inhibition of adenosine uptake into rat brain synaptosomes by the benzodiazepines.

Receptor-mediated noradrenaline stimulation of (Na+-K+) ATPase in rat brain cortical homogenates

Diazepam and flurazepam inhibit adenosine uptake by rat brain synaptosomes.

Adenosine may regulate the vascular supply and thus the growth and spread of neoplastic tissues: A proposal