Constitutive µ-opioid receptor activity leads to long-term endogenous analgesia and dependence.

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced µ-opioid receptor (MOR) constitutive activity (MOR(CA)) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3',5'-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MOR(CA) initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MOR(CA) suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Chronic and acute regulation of Na+/Cl- -dependent neurotransmitter transporters: drugs, substrates, presynaptic receptors, and signaling systems.

Na+/Cl- -dependent neurotransmitter transporters, which constitute a gene superfamily, are crucial for limiting neurotransmitter activity. Thus, it is critical to understand their regulation. This review focuses primarily on the norepinephrine transporter, the dopamine transporter, the serotonin transporter, and the gamma-aminobutyric acid transporter GAT1. Chronic administration of drugs that alter neurotransmitter release or inhibit transporter activity can produce persistent compensatory changes in brain transporter number and activity. However, regulation has not been universally observed. Transient alterations in norepinephrine transporter, dopamine transporter, serotonin transporter, and GAT1 function and/or number occur in response to more acute manipulations, including membrane potential changes, substrate exposure, ethanol exposure, and presynaptic receptor activation/inhibition. In many cases, acute regulation has been shown to result from a rapid redistribution of the transporter between the cell surface and intracellular sites. Second messenger systems involved in this rapid regulation include protein kinases and phosphatases, of which protein kinase C has been the best characterized. These signaling systems share the common characteristic of altering maximal transport velocity and/or cell surface expression, consistent with regulation of transporter trafficking. Although less well characterized, arachidonic acid, reactive oxygen species, and nitric oxide also alter transporter function. In addition to post-translational modifications, cytoskeleton interactions and transporter oligomerization regulate transporter activity and trafficking. Furthermore, promoter regions involved in transporter transcriptional regulation have begun to be identified. Together, these findings suggest that Na+/Cl- -dependent neurotransmitter transporters are regulated both long-term and in a more dynamic manner, thereby providing several distinct mechanisms for altering synaptic neurotransmitter concentrations and neurotransmission.

Protein tyrosine kinase inhibitors alter human dopamine transporter activity in Xenopus oocytes.

The dopamine (DA) transporter (DAT) regulates dopaminergic synaptic transmission by controlling extracellular levels of DA. Thus, understanding signaling mechanisms that alter DAT function is critical for understanding dopaminergic neurotransmission. We have expressed the human DAT (hDAT) in Xenopus laevis oocytes to test the hypothesis that protein tyrosine kinases (PTKs) acutely regulate DAT function by altering cell surface expression of the transporter. Using a relatively high concentration of DA (10 microM), we found that several PTK inhibitors, namely, genistein, lavendustin A, and tyrphostin 25 (10 microM), decreased DA uptake velocity by 58, 41, and 30% of control, respectively. Furthermore, genistein potently inhibited DA uptake with a K(i) = 68 nM. Kinetic analysis confirmed that genistein decreased the V(max) of the DAT, with no change in K(m). The effects of PTK inhibition on hDAT-associated currents were also measured. All three PTK inhibitors attenuated substrate transport-associated currents to similar extents as DA uptake. In contrast, the potent Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) did not significantly inhibit either DA uptake or transport-associated currents. PTK inhibitors decreased hDAT-associated leak currents, however in a more variable manner than for uptake and transport-associated currents. Genistein also decreased cell surface binding of [(3)H]WIN 35,428 to hDAT by 48% of control. Together, these data provide several lines of evidence suggesting that PTK inhibition rapidly reduces hDAT activity via redistribution of the transporter away from the cell surface. Thus, PTKs likely represent another component of cellular signaling cascades that acutely regulate neurotransmitter transporters.

Estradiol modulates vascular response to melatonin in rat caudal artery.

The purpose of this study was to determine whether estrogen modulates the function of vascular melatonin receptors. We used the rat caudal artery and found that the contractile effects of melatonin were influenced by the estrous cycle, ovariectomy, and estrogen replacement. In arterial ring segments isolated from female rats, melatonin potentiated, in a concentration-dependent manner, contractions produced either by adrenergic nerve stimulation or by phenylephrine. Constrictor responses to melatonin were smaller in arteries from female rats in proestrus compared with other stages of the estrous cycle and after ovariectomy. Administration of 17beta-estradiol to ovariectomized female rats also resulted in decreased constriction of isolated arteries to melatonin; however, in vitro addition of 17beta-estradiol (10(-7) M) had no effect. In the caudal artery, melatonin appears to act on two receptor subtypes that mediate contraction and relaxation, respectively. The selective melatonin MT2-receptor antagonist 4-phenyl-2-propionamidotetraline (4P-PDOT) enhanced constrictor responses to melatonin in arterial segments from intact female rats, consistent with the inhibition of MT2 receptor-mediated relaxation. In contrast, 4P-PDOT had no significant effect in arteries from ovariectomized female rats. However, when estradiol was replaced in vivo, the effect of 4P-PDOT on melatonin responses was restored. Thus circulating estradiol appears to enhance MT2 melatonin-receptor function in the thermoregulatory caudal artery of the female rat resulting in increased vasodilatation in response to melatonin.

Melatonin mediates two distinct responses in vascular smooth muscle.

The pineal hormone melatonin was found to produce two distinct contractile responses in vascular smooth muscle. In isolated rat caudal artery segments, denuded of endothelium, melatonin (10(-10)-10(-7) M) potentiated phenylephrine-induced contractions in a concentration-dependent manner. At higher melatonin concentrations (10(-7)-10(-5) M), however, the potentiating effect was attenuated. In the presence of the melatonin MT2 receptor antagonist, 4-phenyl-2-acetamidotetraline (4P-ADOT), the attenuated constrictor responses were selectively enhanced. These results are consistent with the hypothesis that melatonin activates two receptor subtypes in vascular smooth muscle; MT2 receptors may induce relaxation, while a second receptor subtype mediates vasoconstriction.

Ovariectomy eliminates sex differences in rat tail artery response to adrenergic nerve stimulation.

The influence of gonadal hormones on vasoconstrictor responses to adrenergic nerve stimulation was investigated by comparing tail arteries from intact and gonadectomized male and female Fisher 344 rats. Arterial ring segments from females were significantly less responsive to transmural nerve stimulation (1-8 Hz) than arteries from age-matched males. Significant male-female differences persisted after correcting the contractile responses for sex-related differences in arterial mass, optimal resting tension, and maximal contractile force. Arteries were taken from cycling, intact females in either proestrus, estrus, metestrus, or diestrus, but no significant differences were found among the four stages for vasoconstrictor responses to either adrenergic nerve stimulation or exogenous norepinephrine. These data suggest adrenergic function in the artery is not affected by hormonal variations during the estrous cycle. After bilateral ovariectomy, however, contractile responses of female arteries to adrenergic nerve stimulation were increased to levels similar to those observed in male arteries. Orchidectomy of males, in contrast, had no effect on neural-evoked contraction. Low concentrations of norepinephrine also produced greater contractile responses in male compared with female arteries; however, this sex-related difference was eliminated by orchidectomy but not ovariectomy. Taken together, the results indicate that circulating gonadal hormones contribute to gender differences observed in rat tail artery. Vasoconstrictor responses to exogenous norepinephrine appear to be enhanced by testicular hormones. In contrast, vasoconstriction induced by adrenergic nerve stimulation appears to be influenced by chronic exposure to circulating ovarian hormones, resulting in a smaller vascular response in female arteries.