Phenoxybenzamine Selectively and Irreversibly Inactivates Dopaminergic D₂ Receptors on Primary Cultured Rat Lactotrophs

Abstract

Lactotrophs have several different kinds of receptors, such as dopaminergic D₂, somatostatin, angiotensin II and thyrotropin-releasing hormone receptors, which stimulate or inhibit prolaction release. We have studied the specificity of phenoxybenzamine on receptors in lactotrophs. Phenoxybenzamine is a β-haloalkylamine which alkylates chemically active radicals such as hydroxy, sulfhydryl, and amino groups. This alkylation is an irreversible chemical reaction in contrast to the receptor-secretagogue complex which is present in a state of dynamic equilibrium. Primary cultured rat adenohypophyseal cells were used in this study. A dose-response relationship was examined between concentrations of phenoxybenzamine pretreatment and prolactin release using a monolayer cell culture system. The inhibitory action of dopamine (10 µmol/l) on the control group (13.0 ± 0.1 ng/ml or 86% inhibition relative to the control) was significantly higher than on the 0.1-µmol/l phenoxybenzamine-pretreated group (39.0 ± 0.2 ng/ml or 58% inhibition relative to the control), but the stimulatory effect of thyrotropin-releasing hormone on prolactin release was not significantly affected up to a 10-µmol/l phenoxybenzamine pretreatement as compared with the control group. We thus selected a phenoxybenzamine concentration of 0.1 µmol/l for the next series of perifusion experiments in order to examine dynamic changes in prolactin release. The basal prolaction release was decreased to almost half by phenoxybenzamine pretreatment. The inhibitory action of dopamine (0.1 µmol/l containing 0.1 mmol/l ascorbic acid) was significantly less in the phenoxybenzamine-pretreated group (68% of the basal prolactin concentration) than in the control group (31% of the basal concentration). However, the relative actions of somatostatin, thyrotropin-releasing hormone, angiotensin II, and dibutyryl adenosine 3’,5’-cyclic adenosine monophosphate were not significantly affected by the phenoxybenzamine pretreatment. These observations suggest that phenoxybenzamine is relatively selective to dopaminergic D₂ receptors in primary cultured lactotrophs. The basal prolactin release in the phenoxybenzamine-pretreated group increased steadily during the experimental period. The basal prolactin release was doubled within 153.7 ± 22.3 min.

Activation of the Adrenergic β-Receptor Stimulates Prolactin Release from Primary Cultured Pituitary Cells

“…therefore, inactivated the do paminergic Di-receptors with phenoxybenzamine prior to studying the stimulatory action of catecholamines on prolactin release. Phenoxybenzamine was chosen since it blocks irreversibly and its half-recovery time is greater than 2 h [13]. Competitive antagonism, on the other hand, allows the receptors to return to normal function immediately after removal of the antagonist.…”

“…Since phenoxybenzamine inactivates both the a-adrenergic receptors and the Di-receptors [13], and since epinephrine, an a-|b and D:-receptor agonist, stimula tion of prolactin release from phenoxybenzamine pre treated cells likely involves the |breceptor. It is interesting to note that elevated prolactin release induced by isopro terenol, a relatively pure |breceptor agonist, returned to a basal concentration during the isoproterenol perifusion period in nonphenoxybenzamine-treated control groups, but pretreatment with phenoxybenzamine enhanced the stimulatory effect of isoproterenol on prolactin release for a longer period of time.…”

“…Although dopamine pre dominantly inhibits prolactin release, at low concentra tions dopamine stimulates its release [9-1 1 ]. This stimula tory action of dopamine on prolactin release suggests the presence of highly sensitive catecholamine receptors in addition to the excitatory Dj-receptor subtype [11], Fol lowing pretreatment with phenoxybenzamine, an agent that blocks both adrenergic a-receptors [12] and dopami nergic Dj-receptors [13] while keeping the integrity of the adrenergic (1-receptors [ 14], the stimulatory action of cate cholamines on prolactin release was tested. This pharma cological study reveals the presence of adrenergic p-receptors within lactotrophs, and demonstrates a receptor whose P2-subtype acts to stimulate prolactin secretion.…”

Dopamine agonists both stimulate and inhibit prolactin release in GH4ZR7 cells

“…Exposure of D 2 receptorexpressing cells (e.g. GH 4 C 1 -hD 2s ) to the receptor alkylating compound, phenoxybenzamine, for 1 h could abolish D 2 receptors (38), and we showed that the treatment markedly reduced the DA-mediated inhibition of prolactin release from primary cultured rat pituitary cells while phenoxybenzamine pretreatment was ineffective on somatostatin-, TRH-, and angiotensin-II-induced prolactin release (33). Our observation that phenoxybenzamine inactivates DAinduced prolactin release, is consistent with our prevous studies (33).…”

“…We selected three wellestablished D 2 receptor agonists (dopamine, apomorphine and bromocriptine) to investigate whether the DA-induced stimulatory effect on prolactin secretion in GH 4 ZR 7 cells is unique to DA, or if it is a general phenomenon of other DA agonists. In addition, we used a dopamine D 2 receptor antagonist (haloperidol) and an alkylating agent (phenoxybenzamine) of aadrenergic and DA receptors (33) to confirm that all the observed dopaminergic actions were indeed mediated via activation of dopamine D 2 receptors on the GH 4 ZR 7 cells.…”