In the past receptors were identified from binding studies of selective agonists and selective antagonists. It was Ahlquist in 1948 using a quantitative approach to receptor identification who proposed the existence of two adrenoceptor subtypes, a and b, based on different rank orders of potency, within a series of structurally related natural and synthetic agonists, when responses to these agonists were evaluated in different tissues. His classification was consistent with that based on antagonist sensitivity. This kind of proof for the existence of receptors stands valid today, indeed the existence of a and b receptors is not disputed. However in the last 20 years proof of receptor existence has not totally been dependent on the proposed receptor fulfilling the pharmacological criteria. With the development of molecular biological techniques the situation became more complex, leading to further subdivisions of both a1 and a2 adrenoceptors. If a receptor can be cloned - almost irrespective of binding studies - it will be recognised as a receptor. This conflicting method of classifying a receptor has caused many problems in the study of adrenoceptors.
To date at least 3 a1 adrenoceptor subtypes have been identified pharmacologically, at the same time 3 distinct a1 adrenoceptor subtypes have been identified using molecular biological techniques. Unfortunately, the relationship between the pharmacologically defined subtypes and the cDNA clones, and the nomenclature used to describe them was inconsistent and controversial for years.
Experiments conducted on a adrenoceptors showed differences in potency of the antagonists; prazosin and phenoxybenzamine in functional in vitro assays, this finding was the first implication of multiple a1-adrenoceptor subtypes. Initially the a1 adrenoceptors were divided into the a1A and a1B subclasses based on differential affinity of the competitive antagonists WB4101 and phentolamine, and the site directed alkylating agent, chloroethylclonidine. Phentolamine has a high affinity for a1A and a low affinity for a1B. Chloroethylclonidine selectively alkylates the a1B subtype, and not the a1A subtype. The evidence for these subclasses is further supported by the identification of several antagonists showing at least 100 fold selectivity for the a1A adrenoceptor, such as 5-methylurapidil and niguldipine.
The a1b receptor (Upper case refers to pharmacologically identified receptors, e.g. a1B, lower case refers to cloned receptors e.g. a1b) was the first to be cloned. The expression of this cDNA resulted in a protein with high affinity for prazosin and a low affinity for phentolamine, 5-methylurapidil and yohimbine. It was also sensitive to irreversible inactivation by chloroethylclonidine. These radioligand binding properties were consistent with those for the pharmacologically identified a1B adrenoceptor. Therefore the recombinant and the pharmacologically defined receptors were the same receptor. This picture of straightforward identification of the adrenoceptor subtypes did not continue.
By using the cloned a1b adrenoceptor as a probe the discovery of more adrenoceptor subtypes occurred. It was through this method that the now called a1d was found. The new clone was shown to be a seven transmembrane spanning G-protein-linked receptor. Northern analysis of the tissue distribution of mRNA transcribed by this clone showed a similar distribution to that of the a1A adrenoceptor. The expression of this cDNA resulted in a protein that had a high affinity for WB4101 which was also consistent with the a1A adrenoceptor (Lomasney et al 1991b). Unfortunately, Perez (1991) found evidence against the clone being the a1A adrenoceptor. Whilst studying the clone taken from the same tissue as Lomasney he found the clone to have a low affinity for the more selective a1A adrenoceptor antagonists: 5-methylurapidil and niguldipine. This threw doubt on the identity of the receptor so it was consequently termed the a1a/d adrenoceptor.
Another clone was made from the a1b clone in a similar fashion to that of the a1a/d clone. The protein expressed by this clone is pharmacologically distinct from either the a1b or a1a/d adrenoceptors. Interestingly the new receptor has a pharmacological profile very similar to that of the a1A adrenoceptor, in that it has a relatively high affinity for 5-methylurapidil, and a high affinity for WB4101. Unfortunately the new receptor also showed high sensitivity to chloroethylclonidine, which is an a1b like property. The receptor was therefore labelled a1c. Lately the discrepancy has been explained by differences in experimental conditions, the character of the membrane in which the receptor is expressed, and/or species difference. As a consequence of the similarities between a1c and a1A the recombinant receptor is now termed a1a.
Since a1c is now known as a1a, a1a/d is now referred to as a1d. The subdivision of the a1 adrenoceptors, with both pharmacologically native and recombinant cloned subtypes is summarised in the following table.
|Native||Cloned (new nomenclature)||Cloned (old nomenclature)||Human chromosome location|
The work done to identify subtypes of the a2-adrenoceptors has led to the discovery of a greater number of subtypes than a1 receptors, despite this the overall picture is much clearer. As with the a1 adrenoceptors scientists have identified new subtypes both pharmacologically, through radioligand binding studies, and by molecular biological cloning techniques. The subclassification of a2- adrenoceptors was initially based on the ability of prazosin to inhibit the binding of [3H] yohimbine or [3H] rauwolscine to tissue homogenates. Studies comparing human platelet and neonatal rat lung showed each tissue to have a homogenous a2- adrenoceptor population. The ability for prazosin and another antagonist ARC 239, to inhibit [3H] rauwolscine or [3H] yohimbine binding was very different for the two tissues. Prazosin and ARC 239 have high affinity for one group of a2 adrenoceptors found on neonatal rat lung, designated a2B, and low affinity for another found on human platelets, designated a2A.
This evidence coupled with other antagonists and some partial agonists that showed differential selectivity has confirmed the existence of the a2A and the a2B receptors. Interestingly the physiological catecholamines: adrenaline and noradrenaline do not show selectivity over these receptors and will therefore bind unpreferentially to either a2A or a2B.
By the correlation of the affinities for a series of a-adrenoceptor antagonists as inhibitors of [3H] rauwolscine binding in different tissues, two new a2-adrenoceptor subtypes have been identified. The a2C-adrenoceptor is very similar to the a2B-adrenoceptor, having a relatively high affinity for prazosin, ARC 239, and spiroxatrine, but it has a higher affinity for rauwolscine. This may be a subtle difference in pharmacology but the two receptors also differ in the selectivity of certain antagonists. a2C adrenoceptor has a number of selective antagonists including: BAM 1303, and WB 4101 these ligands show no selectivity at the a2B-adrenoceptor.
Another subtype, a2D has also been identified. a2D has a lower affinity for [3H] rauwolscine than the other subtypes and, like the a2A adrenoceptor a low affinity for prazosin, spiroxatrine and ARC 239. Other than yohimbine and rauwolscine, only BAM 1303 has moderate selectivity between a2D- and a2A- adrenoceptors, but the two subtypes can be distinguished when the potency ratios for several antagonist pairs are compared. Other studies in different tissues have caused confusion between these subtypes, but it is now clear that the a2D-adrenoceptor is the rat homolog of the human a2A- adrenoceptor.
The work done on recombinant a2- adrenoceptors has led to the expression of 9 clones. 3 clones from humans, 3 clones from the rat, and 3 clones from the mouse. The first a2-adrenoceptor to be cloned was a2C10 named because it was located on human chromosome C10. The receptors that were later discovered were named in a similar fashion. It is easier to summarise the relationship between the pharmacologically defined recombinant a2 adrenoceptor subtypes in a table.
Lands and his co-workers first identified the existence of two b adrenoceptor subtypes back in 1967. b1 and b2 were originally distinguished by rank order of potency of a series of endogenous and synthetic agonist. The b1 and b2 adrenoceptor sub-classifications are further supported by the development of subtype selective agonists and antagonists.
Evidence has now accumulated throughout the years for the existence of a b adrenoceptor that is insensitive to commonly used antagonists. In the past this receptor was referred to as the "atypical b adrenoceptor", but with the identification of selective agonists, and the expression of a recombinant receptor having similar characteristics, it is now appropriate to refer to this receptor as the b3 adrenoceptor.
All of the b adrenoceptors identified in pharmacological studies have been recombinant and expressed. The pharmacological characteristics of the recombinant receptors appear to correspond well with those of the three subtypes identified in native tissues, although there are some reported differences in the case of the b3 adrenoceptor.
All three b subtypes can be activated by noradrenaline and adrenaline. However, in contrast to the a adrenoceptors, the endogenous catecholamines do have differential affinity for the b subtypes. The primary distinction between b1 and b2 adrenoceptors is the relative potencies of adrenaline and noradrenaline, with the two catecholamines being equipotent at the b1 adrenoceptor and adrenaline having a much higher selectivity for the b2 receptor. Conversely, noradrenaline is more potent than adrenaline as a b3-adrenoceptor agonist. The synthetic catecholamine, isoprenaline, is a potent agonist at all b adrenoceptors subtypes. Propranolol and its many analogues are potent antagonists at b1 and b2 adrenoceptors; however, b3 adrenoceptor-mediated responses are much less sensitive to these antagonists.
Both b1 and b2 adrenoceptors can be radiolabelled with [3H]dihydroalprenolol or [125I]iodopindolol and its analogues. While the location of b3 adrenoceptors can be identified using [125I]iodocyanopindolol. All three b adrenoceptor subtypes activate adenylyl cyclase as a primary mechanism for signal transduction.
The development of new techniques at first cast confusion over the classification of these receptors, however as research progressed with these new tools the confusion has gradually lifted. It seems clear that the slight differences in pharmacological characteristics of many of the subtypes is often due to species differences. Further studies hopefully will not complicate the subject further, but will instead serve to clear up any uncertainties.