A photodynamic effect is produced whenever an isolated rabbit thoracic aorta, which has been previously contracted to a steady-state isometric tension by an aadrenoceptor agonist, is exposed to ultraviolet (UV) light [1,2]. The drug-induced isometric tension rapidly decreases, remains low during the duration of exposure to UV light, and spontaneously returns to baseline tension upon removal of the UV light. The phenomenon occurs independently of the agonist used, but in agonist-specific pharmacodynamic manner. We (RJT, RBR) have previously published evidence suggesting that the photorelaxation is due to the disruption of ligand-receptor bonds [3-6]. Some of the evidence includes (see the review [4]): comparison of agonist dissociation constants [7]; the use of selective receptor antagonists; potentiation of g aminobutyric acid (GABA)-induced current mediated by a1b2g2 GABA receptors [8]; and a decreased number of exposed antigenic determinants, which do not contain photoreactive amino acid residues, on bacteriophage MS2 [9].

We previously published on the utility of planarians for investigating drug action and physiological processes involved in drug abuse [10]. Planarians have a simplified nervous system and mammalian-like neurotransmitter systems, such as dopamine (e.g., [11,12]), acetylcholine [11,12], and opioid [13]. Planarians respond with doserelated behavioral changes to drug exposure or to withdrawal. As an example, dopamine D1- and D2-receptor agonists, antagonists, or inhibitors of neuronal dopamine reuptake alter planarian locomotor activity (“motility”) (e.g., [11,12,14-18]; anticonvulsants inhibit seizure-like activity (pSLA) [19]; and abstinenceand antagonistinduced withdrawal signs are elicited in planarian model of physical dependence [20-23]. Attenuation of these effects by receptor-selective antagonists supports recaptor-mediated mechanisms.

We have shown that a decrease in planarian spontaneous locomotor velocity (pLMV) produced by the selective dopamine D2 receptor antagonist sulpiride (5-(Aminosufonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy benzamide) occurs in an enantiomeric-selective-((–)sulpiride >> (+)sulpiride) and in a dose-dependent manner [24], and that the effect is attenuated by UV light [25]. Amphetamine produces the opposite effect on pLMV (i.e., increases), and the increase is also attenuated by UV light [18]. That the UV light disrupts opposite drug-induced effects on pLMV is consistent with an action at receptors or transduction pathways. If true, then UV light should affect a variety of receptor-mediated effects. The purpose of the present study was to extend and challenge this hypothesis by determining if UV light would attenuate diverse endpoints––viz., pLMV [24], pSLA [19], and physical dependence (withdrawal signs) [20,21]––produced by multiple diverse drugs––viz., (–)sulpiride, nicotine, pilocarpine, and U-50,488H (trans-(±)-3,4-Dichlro-Nmethyl-N-(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide).

The phenomenon of UV light-induced photorelaxation in mammalian isolated tissue preparations has been reviewed previously [4]. Briefly, a strip or ring of rabbit thoracic aorta in state of active contraction induced by norepinephrine or other a-adrenoceptor agonist will exhibit a rapid decrease in the agonist-induced tension when exposed to UV light of sufficient intensity [1,2,5]. The photorelaxation occurs within 1 sec and is reversible, that is, tension is restored to pre-radiation levels upon cessation of the UV light exposure. A series of studies using this preparation has led to the proposal that the energetics of UV light disrupts drug-receptor binding equilibrium [27]. Some of the evidence in support of this includes: values of agonist-receptor dissociation constants determined by photorelaxation agree with corresponding values determined by other method [5]; photorelaxation kinetics of agonists in the presence of rcversiblc and irreversible antagonists are in agreement with theoretical predictions [3,28]; a-adrenoceptor antagonists do not alter the time constant of agonists at other (e.g., angiotensin) receptors [3]. To test the possibility that UV light induces the synthesis of an agonistselective ‘relaxing’ substance, two muscle chambers, each containing a norepinephrine-contracted aorta strip, were aligned vertically, such that the bathing solution of the lower chamber could be quickly replaced by that of the upper chamber. The strip in the upper chamber was exposed to UV radiation, producing photorelaxation––no effect was produced on the strip in the lower chamber. We also tested if UV light degrades ligand by exposing norepinephrine to UV light for 10 min, then testing its ability to contract aorta strips. There was no difference in the magnitude of response (tension development) [4].

To further test the concept that the UV light photorelaxation phenomenon is intimate to the ligandreceptor interaction or transduction, we speculated that it should be manifested by a variety of diverse drugs acting at different receptors and producing different effects. This is difficult to achieve using mammalian models, so we chose to use an invertebrate model with which we have extensive experience [10]. We selected four drugs: three of them agonists (nicotine, U-50,488H, and pilocarpine) and––in order to examine the influence of the absence of signal transduction processes––an antagonist ((–)sulpiride). The four drugs act at different receptors: dopamine D2 (DAR2), nicotinic cholinergic (nAChR), muscarinic cholinergic (mAChR), and k-opioid (KOR). Three of the receptors are metabotropic, namely, G protein-coupled (GPCRs), and the other one is ionotropic (i.e., nAChR). Three diverse drug-induced quantifiable endpoints were selected for study: spontaneous locomotor activity, seizures, abstinence-induced withdrawal (physical dependence).

Dopamine has been identified in planarians [12,14]. It is present in relatively high concentrations [29], and it is localized to dense-core vesicles in synapses [30]. Dopamine receptor agonists increase, dopamine receptor antagonis decrease cAMP levels in planarians [11,15]. Agonist-induced behavioral effects are mediated through dopamine D1- and D2-like receptors and are attenuated by selective receptor antagonists [15]. Regarding the endpoint used in this study planarian locomotor activity, an association between changes in and dopamine receptors has been reported by several groups (e.g., [11,12, 14-17]), and dopamine agonist-induced changes on locomotor activity are antagonized by dopamine receptor antagonist haloperidol [14,17]. Acetylcholine has been identified in planarians [12,14] and behavioral effects are elicited by cholinergic agonists and antagonists and acetylcholinesterase inhibitors [29,31]. The presence of endogenous opioids in planarians has been demonstrated [13], opioid ligands elicit behavioral effects [16] antagonized by the opioid receptor antagonist naloxone and the selective k-opioid receptor antagonist Nor-BNI (Norbinaltorphimine) [14]. Physical dependence development and abstinence-induced withdrwal from U-50,488H has been described previously [21]. Seizure induction in planarians has also been described previously [19].

We demonstrated previously [25] that (–)sulpiride decreases pLMV in an enantiomer-specific manner and the effect is attenuated by UV light. This was duplicated in the present study, which demonstrated that the effect of UV light is reversible. Since amphetamine-induced increase in pLMV is also attenuated by UV light [18], the effect of UV light on this endpoint does not appear to be at the level of 2^nd-messenger transduction pathways, but rather at the level of the receptor. We demonstrated that UV light attenuation of the amphetamine-induced effect is not due to degradation of amphetamine by running a spectrophotometric scan (model DR/4000U, HACH Co., Loveland, CO) over 10-min exposure to UV light. There was no decrement in peak height or area and no new peak appeared (that would have been indicative of a breakdown product) [18]. Saline was used to provide a control for osmolarity. Neither saline (0.1 mM) during treatment nor saline (0.1 mM) during testing had effect on pLMV [21]. Involvement of opioid receptors in U-50, 488H-induced effect in planarians was demonstrated in prior work by a dose-related naloxone-sensitivity and the specific involvement of k-opioid receptors in physical dependence development was demonstrated in planarians that were co-incubated with nor-BNI together with U-50,488H and then tested in water displayed pLMV that was not different from naïve planarians [21]. The pH of solutions containing same concentrations of U-50,488H or naloxone were not different from the pH of the test water [21].

In summary, the spontaneous locomotor activity of planarians was decreased by (–)-sulpiride and was reverted back toward the control levels when the planarians were exposed to UV light; seizures were induced in planarians by nicotine and pilocarpine and were attenuated when the planarians were exposed to UV light; and development of physical dependence to U-50,488H (manifested by abstinence-induced withdrawal) was attenuated by UV light. The observed effects of UV light cannot be attributed to toxic effects, since the UV light had no effect of its own on the behaviors. The observed effects of UV light have also been shown to attenuate antagonist—as well as agonist-induced effects—which argues against an effect of UV light only on ligands having intrinsic activity (efficacy). The results also argue against UV light releasing some endogenous substance, since UV light counteracts opposite effects. Based on the demonstration in the present study of attenuation by UV light of four different ligands selective for four different receptors and at three different endpoints, the results are compatible with the view that UV light disrupts ligand-receptor binding.

The authors thank Timothy Shickley, Ph.D., for suggesting Planaria as a model. This work was supported by NIDA grants DA15378 (RBR) and DA022694 (SMR).