"Caged" phenylephrine: development and application to probe the mechanism of alpha-receptor-mediated vasoconstriction.

Abstract

A "caged" analogue of the a-adrenergic receptor agonist phenylephrine (PE) was prepared by exploiting the 2-nitrobenzyl protecting group and using a synthetic procedure developed to permit preferential derivatization at the amino group. On isolated adult rat mesenteric arterioles, caged-PE had no measurable effects at concentrations up to 100 IAM; 0.5-ms light flashes in the presence of caged-PE, however, produced marked and dose-dependent vasoconstriction. Flashinduced vasoconstrictions were blocked by the a-receptor antagonist phentolamine and were unaffected by the 13-receptor antagonist propranolol, indicating that the light-induced responses reflect the selective activation of a-adrenergic receptors. After a single flash, a large transient decrease in vessel diameter was recorded, and in most vessels, this was followed by a smaDler, sustained constriction. The sustained component of the contraction was selectively eliminated when Ca2+ was removed from the bath, which suggests that different mechanisms underlie the transient and the sustained responses to PE. The responses to single flashes of varying intensities occurred with a mean latency of 460 ms, which is consistent with the intermediacy of several steps between a-receptor activation and contraction. We anticipate that it will be possible to extend this approach to develop caged analogues of other neurotransmitters for mechanistic and kinetic studies.Previously, this laboratory and others (1-3) have exploited the 2-nitrobenzyl protecting group (see Fig. 1) in the development of "caged" analogues of a variety of intracellular second messengers. The rationale for this approach was the anticipated spatial and temporal resolution afforded by controlling the intracellular messenger application with light, as compared to more conventional techniques. There are, however, a number of criteria that a caged compound need fulfill to be useful in biological experiments (1-3). First, the caged analogue should lack biological activity, so that it can be applied under steady-state conditions. Second, photorelease should proceed with reasonable quantum efficiency and be devoid of competing or side reactions. Third, photorelease should be fast and irreversible, preferably being complete in '1 ms. Finally, any side products produced on photolysis should be chemically inert, biologically inactive, and nontoxic. Given the success of this approach in providing caged intracellular second messengers (1-3) and our interest in the mechanisms involved in the modulation of voltage-gated ion channels by adrenergic receptor stimulation (4-6), we reasoned that this methodology could be exploited to develop caged adrenergic receptor agonists. In support of the general applicability of this approach, the 2-nitrobenzyl moiety has recently been used in the development of caged analogues of carbamylcholine, a nicotinic cholinergic receptor agonist (7,8).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marke...

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“…A long lag phase between the binding of an excitatory agonist to its receptor and contraction is a characteristic property of smooth muscle, evident even when diffusional delays are eliminated through photolysis of caged agonists (Somlyo et al, 1988a;Muralidharan et al, 1993;Walker et al, 1993). A major component of the delay (1-2 s in guinea pig portal vein smooth muscle stimulated with phenylephrine at room temperature; Somlyo et al (1988a)) is due to phospholipase C-mediated generation of inositol 1,4,5-trisphosphate that is the messenger of pharmacomechanical Ca 2ϩ release from the sarcoplasmic reticulum (reviewed by Somlyo and Somlyo (1990)).…”

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Investigating the degradation of the sympathomimetic drug phenylephrine by electrospray ionisation-mass spectrometry

“…Another approach was the synthesis and characterisation of bioreversible phenylephrone derivatives [42]. The preparation of a "caged" phenylephrine as a product of selective derivatization on the amine function was an approach to probe the mechanism of ␣ receptor mediated vasoconstriction in isolated adult rat mesenteric arterioles [43].…”