0014-4754/93/040329-0351.50 + 0.20 ~) Birkhfiuser Verlag Basel, 1993 329 Potentiating effect of phenylephrine on isoproterenol activation of thyroxine type II deiodinase in the pineal gland of adult rats C. Osuna, A. Rubio and J. M. Guerrero* The University of Seville, School of Medicine, Department of Medical Biochemistry and Molecular Biology, Avda Sanchez Pizjuan 4, E-41009 Seville, (Spain) Received 14 December 1992; accepted 15 January 1993 Abstract. In the present study we show, for the first time, that phenylephrine (PHE), an c~-adrenergic receptor agonist, potentiates the effect of isoproterenol (ISO), a fi-adrenergic agonist, in activating pineal type II5'-deiodi- nase (5'-D) activity. The potentiating effect of PHE was observed only at doses of ISO which induce submaximal activation of the enzyme. However, at doses which lead to maximal activation of the enzyme, PHE was ineffective. The results suggest that not only fi-, but also c~-adrenergic receptors, are involved in the sympathetic noradrenergic regulation of pineal 5'-D activity in the adult rat. Key words'. Pineal; thyroxine; deiodinase; phenylephrine; isoproterenol. Thyroxine type lI5'-deiodinase (5'-D) converts the rela- tively inactive thyroid hormone T4 into its highly active metabolite, T3. This enzyme is present in specific tissues including anterior pituitary, brain, brown adipose tis- sue, epidermal keratinocytes, the Harderian gland, and the pineal gland ~. The most important regulatory mech- anism for this isoenzyme is the thyroid status. There is a significant increase in its activity during hypothy- roidism, and a marked inhibition in the presence of T42'3. In the rat pineal gland, 5'-D activity is regulated by the light:dark cycle as well as by the thyroid status. The enzyme exhibits a progressive rise in activity alter the onset of the dark period and reaches a peak value 5 6 h later; this peak coincides with the peak values described for both melatonin content and N-acetyl- transferase (NAT) activity 4 ~. This nocturnal increase in 5'-D activity seems to be dependent on the sympa- thetic noradrenergic input, since either continuous light exposure or superior cervical ganglionectomy prevents it 5. Additionally, both in vivo and in vitro studies have shown that isoproterenol (ISO), a fl-adrenergic agonist, also activates 5'-D activity, while propranolol, a fi- adrenergic blocker, inhibits it ~' m In the present study we show for the first time that phenylephrine (PHE), an ~-adrenergic agonist, potenti- ates the effect of ISO, which is known to be a potent activator of pineal 5'-D activity. The results suggest that not only fl-, but also c~-adrenergic receptors are in- volved in the sympathetic noradrenergic regulation of pineal 5'-D activity in the adult rat. Materials and methods Male Wistar rats, weighing 100-120 g at the time of the study, were allowed to acclimatise to the animal facili- ties. Animals received food and water ad libitum and were exposed to an automatically regulated light:dark (LD) cycle of 14:10; the lights were turned off daily from 20.00 h to 06.00 h. On the day of the experiment, animals were subcutaneously injected with ISO or/and PHE at 12.00h and 14.00h, and killed at 16.00h. Control animals were injected with saline. Pineals were quickly collected and stored at -80 ~C for 5'-D deter- minations. The measurement of 5'-D activity was based on the release of radioiodine from [3',5'-125I]T4 . This method, commonly used in pineal gland deiodinating studies 4 to is specific for type lI isoenzyme because the substrate contains ~e5I only in the position 5'. Other deiodinating activities, e.g. conversion of T 4 to rT~, would release only nonradioactive iodide 4. Pineals were disrupted by ultrasound in 100 tal cold 0.05 M phosphate buffer, pH 6.8. Briefly, 50 lal of a sonicated sample was incubated in the presence of 40 Mm DTT and 2 nM [3',5'-125I]T4 as substrate (200 lal final volume). The substrate con- centration was similar to the K m value described for the type II5'-D activity in rat pineal gland 4. Reaction was started by the addition of substrate and continued for 60 rain at 37 "C. Control incubations were performed by omission of the homogenates. The reaction was terminated by the addition of 100 lal cold 2% BSA and 750 lal trichloroacetic acid (10%). Samples were cen- trifuged for 2 min at 10,000 x g and 500 btl of super- natant was placed on a 0.5 ml column packed with Dowex-50W ion-exchange resin and the column washed twice with 500 I~1 glacial acetic acid (10%). Radioactiv- ity in the eluate, corresponding to the 125I released, was counted in a gamma counter as an index of 5'-D activ- ity. The recovery of 125I in this process was better than 95%. Specific enzymatic activity was determined by sub- tracting the control value, which usually amounted to less than 1% of the radioactivity added. 5'-D activity is referred to as I25I released (fmol/gland/h). Results are