Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence.

MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re-challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin-treated cells. In addition, microtubule depolymerization increases melatonin-induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2-CHO cells enhances the exchange of GTP on Gi-proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin-induced phase-shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 pm melatonin to phase-shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of beta-lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin-induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.

Dual coupling of MT(1) and MT(2) melatonin receptors to cyclic AMP and phosphoinositide signal transduction cascades and their regulation following melatonin exposure.

In this investigation, we wanted to determine whether MT(1) or MT(2) melatonin receptors are capable of coupling to the phosphoinositide (PI) signal transduction cascade. In addition, we wanted to assess the effects of chronic melatonin exposure on MT(1) and MT(2) melatonin receptor-mediated stimulation of PI hydrolysis. We also assessed the effects of chronic melatonin exposure on other parameters of the MT(2) melatonin receptor function including total specific 2-[125I]-iodomelatonin binding, the affinity of melatonin for the receptor, and melatonin (1nM)-mediated inhibition of cyclic 3',5'-adenosine monophosphate (cAMP) accumulation. Investigation of the PI signal transduction cascade activated by either the MT(1) or MT(2) melatonin receptor expressed in Chinese hamster ovary (CHO) cells showed that melatonin (1pM to 1mM) was able to stimulate the formation of PIs to approximately 40-60% over basal [EC(50): MT(1)=29nM (2-300nM) and MT(2)=1.1nM (0.32-3.5nM), N=5]. This response was mediated via receptors based upon the findings that melatonin did not stimulate the formation of PIs in CHO cells devoid of receptor and that antagonism of MT(2) melatonin receptors by 4P-PDOT (AH 024; 4-phenyl-2-propionamidotetralin) attenuated melatonin-mediated stimulation of PI hydrolysis in CHO cells expressing the MT(2) melatonin receptor. The consequence of chronic melatonin exposure on MT(1) and MT(2) receptor function was also examined. Pretreatment of either MT(1)- or MT(2)-CHO cells with melatonin (1 microM for 5hr) resulted in: (a) a complete loss of melatonin-mediated stimulation of PI hydrolysis, and (b) an attenuation of melatonin (1nM)-mediated inhibition of forskolin-induced cAMP accumulation by approximately 20-40%. The desensitization of the PI hydrolysis signal transduction cascades coupled to either MT(1) or MT(2) melatonin receptors following chronic melatonin exposure was not due to depleted phospholipid pools, to elevated basal levels, or to decreases in receptor affinity and density. This dual coupling of melatonin receptors to different signal transduction cascades may contribute to the diversity of melatonin receptor function in vivo.

Modulation of melatonin receptors and G-protein function by microtubules.

Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin-induced outgrowths in MT1-expressing CHO cells, also prevents MT1 receptor desensitization by affecting G(alpha)-GTP exchange on G-proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin-induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin-treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G-proteins. Depolymerization of microtubules during melatonin-induced desensitization, attenuated forskolin-induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of G(i alpha) protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into G(i) proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/G(q) coupling nor did it affect melatonin-induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin-induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on G(i) proteins and impact on downstream signaling cascades.