The inset shows the results with and without morphine for every animal. calcium imaging. Morphine induced a dose-dependent rise in intracellular calcium in neurons from wild-type animals. In neurons from GsMTx4 TRPV1 and TRPA1 knockout animals activation by morphine was markedly reduced, in the TRPV1/A1 double knockout animals this morphine effect was abrogated. Naloxone induced an increase in calcium levels much like morphine. The responses to both morphine and naloxone were sensitized by bradykinin. Conclusion Nociceptor activation and sensitization by morphine is usually conveyed by TRPV1 and TRPA1. Background After many thousands of years of therapeutic opium use, in 1806 the German pharmacist Sertuerner published the discovery of the main pharmacological compound of opium, which he later called morphine . From this time morphine and its derivatives, which form the class of opioids, became the main therapeutic option for the treatment of moderate to severe pain. The analgesia as well as most side Rabbit Polyclonal to XRCC5 effects are caused by interaction with the opioid receptors, especially the mu-receptors. Opioids have several generally known side effects including dependency, sedation, constipation and nausea . Besides their well-known antinociceptive actions, opioids can cause hyperalgesia by an unknown, opioid receptor-independent mechanism [3,4]. This present study was initiated by the clinical observation that in patients suffering from radiodermatitis, which could not be controlled by systemic morphine, high doses of a custom-made opioid gel applied topically to the skin can elicit severe burning pain for a few seconds (Clinical research group KFO130, Erlangen, Germany). The isotonic gel prepared by the university’s pharmacy contains 2.67 mM (0.1%) morphine and has a neutral pH. Case reports have previously reported hyperalgesia after application of high doses of morphine [5,6]. At lesser concentrations morphine only causes pruritus, which might be explained by mast cell degranulation . The transient receptor potential channel TRPV1 (formerly VR1) receptor is found in small to medium diameter dorsal root, trigeminal and nodose ganglia main afferent neurons which sense potentially damaging stimuli such as capsaicin, warmth and low pH [8,9]. For TPRV1 more pungent activating compounds have been reported than for any other TRPV channel. Another TRP channel, TRPA1 (formerly ANKTM1) equips neurons with a sensitivity for mustard oil and many diverse pungent chemical stimuli . Among these compounds are electrophilic compounds such as allyl isothiocyanate, acrolein, formalin, 15-deoxy-12,14-prostaglandin J2, nitric oxide, hydrogen peroxide as well as the inflammatory mediator bradykinin . TRPA1 and TRPV1 are the principal detectors for painful chemical stimuli. The aim of this study was to investigate whether the painful sensations evoked by morphine are mediated by these two receptors. Two models of nociceptive activation were employed using mice lacking TRPV1, TRPA1 or both these receptors. Results Morphine activates hTRPV1 and TRPA1 expressed in HEK293t In HEK293t cells transfected with hTRPV1 application of 10 mM morphine evoked an inward current, 300 M capsaicin was applied as a control thereafter (n = 10, sample recording in Fig. ?Fig.1).1). In HEK293t cells transfected with mTRPA1 10 mM morphine evoked inward currents that appeared to rapidly inactivate during continued superfusion (n = 10). However, upon offset and washout of morphine regularly large tail currents occurred that revealed the sustained activation of TRPA1 and a slow deactivation. Mustard oil (30 M) was applied as a control GsMTx4 thereafter. Open in a separate windows Physique 1 Morphine activates TRPV1 and TRPA1 expressed in HEK cells. HEK cells transfected with cDNA for hTRPV1 and mTRPA1 were exposed to 10 mM GsMTx4 morphine. The upper traces show an inward current in a cell transfected by hTRPV1 and GFP, evoked by 10 mM morphine and by 300 nM capsaicin, both applied for a period of 10 s. The lower traces show GsMTx4 an inward current in a cell transfected by mTRPA1 and GFP, evoked by 10 mM morphine and by 30 M mustard oil, both applied for a period of 40 s. Cells were exposed to 1 M m-3M3FBS before application of morphine and between applications. Morphine elicits calcium transients in DRG neurons A total of 106 dorsal root ganglion (DRG) neurons were exposed to 10 mM morphine for 40 s, 30 M acrolein for 30 s, 1 M capsaicin for 5 s and 60 mM potassium for 30 s with an interval of 240 s between applications. Response rates GsMTx4 to morphine were 22%, to acrolein 45% and to capsaicin 58%. Morphine responses in neurons activated by capsaicin but not by acrolein and in neurons activated by acrolein but not capsaicin.