Potent alpha(2A)-adrenoceptor-mediated vasoconstriction by brimonidine in porcine ciliary arteries.

PURPOSE: An investigation into whether alpha(2)-adrenoceptor agonists induce contractions in the porcine ciliary arteries and to characterize the functional receptor subtype mediating these responses. METHODS: Isolated arteries from the intraocular part of the porcine ciliary artery were suspended in microvascular myographs for isometric tension recording. The segments were contracted with the alpha(2)-adrenoceptor agonists brimonidine, apraclonidine, and oxymetazoline. To determine which subtypes of the alpha(2)-adrenoceptor mediate this contraction, antagonists subselective for the different alpha(2)-adrenoceptors were added to the vessel bath before concentration-response curves for brimonidine were obtained. The following alpha(2)-adrenoceptor antagonists were applied: BRL44408 (alpha(2A)-selective), ARC239 (alpha(2B)- and alpha(2C)-selective), and prazosin (alpha(2B)- and alpha(2C)-selective). RESULTS: The alpha(2)-adrenoceptor agonists induced vasoconstriction in the porcine ciliary artery with the following potency order (EC(50)) expressed in nanomolar: brimonidine 2.11, oxymetazoline 5.26, and apraclonidine 13.0. As a reference, noradrenaline was tested, and its EC(50) was determined to be 247 nM in the ciliary artery. In the porcine ciliary arteries BRL44408, ARC239, and prazosin caused concentration-dependent and parallel rightward shifts of the concentration-response curves for brimonidine. Schild analyses for the antagonists against brimonidine yielded regression lines with slopes of unity and functional antagonist potencies (pK(B)) for BRL44408 (7.8), ARC 239 (5.8) and for prazosin (6.0) suggesting the presence of functional alpha(2A)-adrenoceptors. Moreover, there was a good correlation of pK(B) with ligand-binding affinity (pK(i)) of the alpha(2A)-adrenoceptor in the porcine eye tissue. CONCLUSIONS: The alpha(2)-adrenoceptor agonists brimonidine, apraclonidine, and oxymetazoline are potent vasoconstrictors in the porcine ciliary artery. In the present work, it was shown for the first time that the alpha(2A)-adrenoceptor subtype mediates this contraction.

Characterization of alpha(1)-adrenoceptor subtypes in the eye.

The presence of the alpha(1)-adrenoceptor subtypes in various parts of the pig and rabbit eyes was investigated using [(3)H]-prazosin radioligand binding. The characterization of the subtypes was achieved by performing competition experiments with various subtype selective drugs. In the pig retina, both alpha(1A)- and alpha(1B)-adrenoceptors were detected and the proportion of sites was 70% alpha(1A)- and 30% alpha(1B)-adrenoceptors, respectively. In the pig iris, ciliary body and choroid, which are melanin-rich tissues, the non-specific binding of [(3)H]-prazosin was too high to detect any of the alpha(1)-adrenoceptor subtypes. However, in the albino rabbit iris, ciliary body and retina both alpha(1A)- and alpha(1B)-adrenoceptors were detected. The proportion of sites in the iris was 60 % alpha(1A)- and 40% alpha(1B)-adrenoceptors, respectively. In the ciliary body and rabbit retina the proportion of sites were 70% alpha(1A)- and 30% alpha(1B)-adrenoceptors. Only the alpha(1A)-adrenoceptor subtype was detected in the rabbit choroid.

Characterization of alpha1-adrenoceptor subtypes in the pig.

The identities of the alpha1-adrenoceptor subtypes present in various tissues of the pig were studied using [3H]prazosin radioligand binding. The subtypes were characterized by performing competition experiments for various subtype selective drugs. In the cerebral cortex, spleen and heart, both alpha1A- and alpha1B-adrenoceptors were detected. In the liver was found only the alpha1A-subtype, while in the aorta was found only the alpha1B-subtype. An alpha1-adrenoceptor subtype was present in the adrenal gland with a high affinity for prazosin, the pKd value being 9.6, but with relatively low affinities for other alpha1-adrenoceptor binding drugs. The adrenal gland alpha1-adrenoceptor did not seem to represent the classical alpha1D-subtype, since drugs selective for the alpha1D-subtype in other species, including BMY7378 and SKF104856, showed low affinities for the pig adrenal gland alpha1-adrenoceptor.

[3H]RS79948-197 binding to human, rat, guinea pig and pig alpha2A-, alpha2B- and alpha2C-adrenoceptors. Comparison with MK912, RX821002, rauwolscine and yohimbine.

The Kd values of the recently introduced radioligand [3H]RS79948-197 ((8a R,12aS,13a-S)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-metho xy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) were determined for the recombinant human and rat alpha2A-, alpha2B- and alpha2C- as well as guinea pig alpha2B- and alpha2c-adrenoceptors expressed in COS (CV-1 Origin, SV40) cells. In addition, the Kd values were also determined for [3H]RS79948-197 for the guinea pig spleen alpha2A-adrenoceptor and for pig alpha2A-, alpha2B- and alpha2C-adrenoceptors in membranes obtained from kidney and striatum. Available radioligands for alpha2-adrenoceptors, besides [3H]RS79948-197 are the tritiated forms of MK912 ((2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octa hydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4'-pyrimidin-2'-one), RX821002 (2-methoxy-idazoxan), rauwolscine and yohimbine. In the present article the binding constants of all these substances for the alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes in human, pig, rat and guinea pig are reviewed. In all species tested MK912 was alpha2C-selective, RX821002 showed a minor alpha2A-selectivity, whereas [3H]RS79948-197 was non-selective among the alpha2-adrenoceptor subtypes, showing high affinity for all three subtypes. Rauwolscine and yohimbine showed relatively low affinities for nmost of the alpha2-adrenoceptor subtypes investigated, the exception being rauwolscine having high affinity for the human and porcine alpha2C-adrenoceptors.

Characterization of alpha 2-adrenoceptor subtypes in the porcine eye: identification of alpha 2A-adrenoceptors in the choroid, ciliary body and iris, and alpha 2A- and alpha 2C-adrenoceptors in the retina.

The subtypes of alpha 2-adrenoceptor were characterized in the choroid, ciliary body, iris and retina of the pig eye by using radioligand binding. [3H]-MK912 labelled dense populations of alpha 2A-adrenoceptors in the choroid and ciliary body. In the retina, [3H]-MK912 labelled both alpha 2A- and alpha 2C-adrenoceptors. In the iris, receptors of the alpha 2A-adrenoceptor type were detected by using either [3H]-MK912 or [3H]-RX821002 as radioligands.

Identification of drugs subtype-selective for alpha 2A-, alpha 2B-, and alpha 2C-adrenoceptors in the pig cerebellum and kidney cortex.

The radioligands [3H]MK912 and [3H]RX821002 were used to label alpha2A-, alpha2B-, and alpha2C-adrenoceptors of the pig cerebellum and kidney cortex. By inclusion of the alpha2A-adrenoceptor-selective drug, BRL44408, and using a 'multi-curve' experimental design all the three porcine alpha2-adrenoceptor subtypes could be characterized pharmacologically. The data indicate that the pig alpha2-adrenoceptor subtypes are pharmacologically more related to human alpha2-adrenoceptor subtypes than to the rodent alpha2-adrenoceptors. We suggest a set of drugs that are useful for the delineation of the pig alpha2-adrenoceptor subtypes.