1 A degree of ambiguity and uncertainty exists concerning the distribution of mRNAs encoding the four cloned adenosine receptors. In order to consolidate and extend current understanding in this area, the expression of the adenosine receptors has been examined in the rat by use of in situ hybridisation and the reverse transcription-polymerase chain reaction (RT-PCR). 2 In accordance with earlier studies, in situ hybridisation revealed that the adenosine A1 receptor was widely expressed in the brain, whereas A2A receptor mRNA was restricted to the striatum, nucleus accumbens and olfactory tubercle. In addition, A1 receptor mRNA was detected in large striatal cholinergic interneurones, 26% of these neurones were also found to express the A2A receptor gene. Central levels of mRNAs encoding adenosine A2B and A3 receptors were, however, below the detection limits of in situ hybridisation. 3 The more sensitive technique of RT-PCR was then employed to investigate the distribution of adenosine receptor mRNAs in the central nervous system (CNS) and a wide range of peripheral tissues.As a result, many novel sites of adenosine receptor gene expression were identified. Al receptor expression has now been found in the heart, aorta, liver, kidney, eye and bladder. These observations are largely consistent with previous functional data. A2A receptor mRNA was detected in all brain regions tested, demonstrating that expression of this receptor is not restricted to the basal ganglia. In the periphery A2A receptor mRNA was also found to be more widely distributed than generally recognised. The ubiquitous distribution of the A2B receptor is shown for the first time, A2B mRNA was detected at various levels in all rat tissues studied. Expression of the gene encoding the adenosine A3 receptor was also found to be widespread in the rat, message detected throughout the CNS and in many peripheral tissues. This pattern of expression is similar to that observed in man and sheep, which had previously been perceived to possess distinct patterns of A3 receptor gene expression in comparison to the rat. 4 In summary, this work has comprehensively studied the expression of all the cloned adenosine receptors in the rat, and in so doing, resolves some of the uncertainty over where these receptors might act to control physiological processes mediated by adenosine.

The voltage-sensitive sodium channel confers electrical excitability on neurons, a fundamental property required for higher processes including cognition. The ion-conducting ␣-subunit of the channel is regulated by two known auxiliary subunits, ␤1 and ␤2. We have identified rat and human forms of an additional subunit, ␤3. It is most closely related to ␤1 and is the product of a separate gene localized to human chromosome 11q23.3. When expressed in Xenopus oocytes, ␤3 inactivates sodium channel opening more slowly than ␤1 does. Structural modeling has identified an amino acid residue in the putative ␣-subunit binding site of ␤3 that may play a role in this difference. The expression of ␤3 within the central nervous system differs significantly from ␤1. Our results strongly suggest that ␤3 performs a distinct neurophysiological function.T he voltage-sensitive sodium channel plays a fundamental role in excitable cells, transiently increasing the sodium permeability of the plasma membrane in response to changes in membrane potential and thus propagating the action potential (1, 2). Not surprisingly, mutations in sodium channel genes are implicated in several pathologies, including epilepsy and cardiac arrhythmias (3-5), and therapeutic drugs, including antiepileptics, local anesthetics, and anticonvulsants (6), act on the channel.In the central nervous system, the channel is conventionally described as a heterotrimer composed of a 260-kDa ␣-subunit, a noncovalently associated 36-kDa ␤1-subunit, and a disulfidelinked 33-kDa ␤2-subunit (2). The ␣-subunit forms the ion pore and is responsible for the voltage-sensitive characteristics of the complex. There are multiple isoforms of the ␣-subunit expressed in different regions of the brain and peripheral nervous system that differ in their kinetic properties (1). The ␤-subunits are auxiliary components acting in a regulatory capacity (7). ␤1 increases the fraction of ␣-subunits operating in a fast gating mode, thus accelerating the activation and inactivation kinetics of the channel and modulating the frequency with which neurons fire (8). The ␤2-subunit is required for the efficient assembly of the channel but has minor effects on gating kinetics. These two ␤-subunits are distantly related by sequence (9).We now report the cloning and analysis of the rat and human forms of a previously uncharacterized sequence that we call ␤3. It is homologous to ␤1, but differs from ␤1 both in its distribution within the brain and in some of its kinetic properties. The discovery of this subunit increases the complexity of the sodium channel and raises further questions about the role of these auxiliary subunits. Materials and MethodsCloning Methodology. We isolated a variant of the rat pheochromocytoma cell line PC12 (termed A35C), which lacks typical neuronal properties (10). To discover previously unidentified neuroendocrine-specific genes, subtractive cloning was used to identify transcripts expressed at a level in the variant cells lower than that in normal PC12 cells. Total RNA wa...

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