Susan L. Ingram

Regulation of μ-opioid receptors: Desensitization, phosphorylation, internalization, and tolerance

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance.

Dopamine transporter–mediated conductances increase excitability of midbrain dopamine neurons

Uptake by Na+/Cl−-dependent neurotransmitter transporters is the principal mechanism by which extracellular biogenic amine concentrations are regulated. In addition to uptake, the cloned transporter proteins also elicit ion channel–like currents, but the physiological consequences of these currents are unknown. Here, whole-cell patch clamp and perforated-patch recordings show that substrates of the dopamine transporter (DAT), such as dopamine (DA) and amphetamine, increase the firing activity of rat DA neurons in culture. We found that these substrates elicit inward currents that are Na+-dependent and blocked by cocaine. These currents are primarily comprised of anions and result in an excitatory response in DA neurons at lower DA concentrations than are required for D2 autoreceptor activation. Thus, in addition to clearing extracellular DA, our results suggest that the currents associated with DAT modulate excitability and may regulate release of neurotransmitter from midbrain DA neurons.

Opioid inhibition of Ih via adenylyl cyclase

Opioids are coupled through G proteins to both ion channels and adenylyl cyclase. This study describes opioid modulation of the voltage-dependent cation channel, Ih, in cultured guinea pig nodose ganglion neurons. Forskolin, PGE2, and cAMP analogs shifted the voltage dependence of activation of Ih to more depolarized potentials and increased the inward current at -60 mV. Opioids had no effect on Ih alone, but reversed the effect of forskolin on Ih. This action of opioids was blocked by naloxone. Opioids had no effect on Ih in the presence of cAMP analogs, suggesting that modulation occurs at the level of adenylyl cyclase. The shift in the voltage dependence of Ih by agents that induce inflammation (i.e., PGE2) is one potential mechanism to mediate an increased excitability. Opioid inhibition of adenylyl cyclase and subsequent inhibition of Ih may be a mechanism by which opioids inhibit primary afferent excitability and relieve pain.

Cellular Actions Of Opioids And Other Analgesics: Implications For Synergism In Pain Relief

1. μ‐Opioid receptor agonists mediate their central analgesic effects by actions on neurons within brain regions such as the mid‐brain periaqueductal grey (PAG). Within the PAG, μ‐opioid receptor‐mediated analgesia results from inhibition of GABAergic influences on output projection neurons. We have established that μ‐opioid receptor activation in the PAG causes a presynaptic inhibition of GABA release that is mediated by activation of a voltage‐dependent K+ channel via 12‐lipoxygenase (LOX) metabolites of arachidonic acid.

The Brainstem and Nociceptive Modulation

Spinal nociceptive processing is under the control of specific brainstem modulatory circuits. The best studied and probably functionally most significant such modulatory circuit has links in the midbrain periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). This system exerts bidirectional control over nociceptive processing, suppressing, or facilitating nociception under different physiological and pathological conditions. The PAG–RVM system receives substantial inputs from hypothalamus and forebrain, integrating information related to motivation and emotion with visceral and somatic afferent input. This allows top-down control as well as feedback modulation of nociception. Functional characterization of neurons in the RVM reveals two populations of nociceptive modulatory neurons, on- and off-cells. There is now strong evidence that on-cells facilitate nociception and that off-cells inhibit nociception. A similar circuit-level analysis of the PAG, which is implicated in a host of functions from autonomic control to reproductive behavior and vocalization, would greatly advance our understanding of the neural circuitry of pain modulation in this region. The current challenge is to define the mechanisms through which the PAG–RVM modulatory system is brought into play to enhance or inhibit pain.

Responses of adult human dorsal root ganglion neurons in culture to capsaicin and low pH

&NA; This study examined the responses of cultured adult human dorsal root ganglion (hDRG) neurons to protons and capsaicin, two substances known to produce pain and hyperalgesia in humans. Both substances were applied to each neuron and responses were examined under both voltage‐ and current‐clamp recording conditions. Sensitivity to protons was tested with rapid acidification of the extracellular fluid from pH 7.35 to 6.0. In neurons nominally clamped near −60 mV, low pH evoked a transient inward current which, in all 40 hDRG neurons tested, was followed by a more sustained inward current. The sustained current was associated with an increase in membrane conductance in 10 neurons, a decrease in 27 neurons, and no overt change in conductance (<10%) in 3 neurons. Current‐clamp recordings in the same neurons showed that the proton‐induced sustained net inward current caused a prolonged depolarization of the membrane potential in all 40 hDRG neurons. The prolonged depolarization was associated with action potential discharge in 5 neurons. Unlike low pH, capsaicin evoked a sustained net inward current in only a subset of neurons tested (10 nM: Symbol, 30 nM: Symbol, 100 nM: Symbol, and 10 &mgr;M: Symbol neurons tested). The capsaicin‐evoked currents were accompanied by an increase in membrane conductance in 15 neurons, a decrease in 2, and no overt change in conductance in 9 neurons. Capsaicin currents, like proton‐induced currents, resulted in prolonged depolarizations (10 nM: Symbol, 30 nM: Symbol, 100 nM: Symbol,d and 10 &mgr;M: Symbol neurons tested). The depolarization resulted in the discharge of action potentials in 14 neurons. It is concluded that, while both protons and capsaicin exert excitatory effects on human sensory neurons, multiple membrane mechanisms lead to the depolarization of cultured hDRG neurons by low pH. Inhibition of resting membrane conductances contributes to the responses to low pH in some hDRG neurons. Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available

Antinociceptive tolerance revealed by cumulative intracranial microinjections of morphine into the periaqueductal gray in the rat

The periaqueductal gray (PAG) appears to play a key role in morphine antinociception and tolerance. The objective of this manuscript is to develop a cumulative dose microinjection procedure so the hypothesized role of the PAG in morphine antinociceptive tolerance can be assessed using dose-response analysis. Rats were implanted with a guide cannula into the ventrolateral PAG. Microinjection of cumulative half log doses of morphine (0.32, 1, 3.2, and 10 micro g/0.4 micro l) produced antinociception on the hot plate test only at the two highest doses. Microinjection of quarter log doses of morphine into the PAG (1, 1.8, 3.2, 5.6, and 10 micro g/0.4 micro l) resulted in an ED(50) for antinociception of 1.8 mug. Systemic administration of the opioid antagonist naloxone increased the morphine ED(50) to 9.0 micro g. Repeated microinjections of saline into the PAG had no effect on nociception. Pretreatment with twice daily injections of morphine, either systemically (5 mg/kg, s.c.) or into the PAG (5 micro g/0.4 micro l), for 2 days produced a two-fold increase in the ED(50) for morphine antinociception. These data validate the use of an intracranial cumulative dose procedure to assess morphine potency and demonstrate that microinjection of morphine into the PAG is sufficient to produce tolerance.

Amphetamine Modulates Excitatory Neurotransmission through Endocytosis of the Glutamate Transporter EAAT3 in Dopamine Neurons

Amphetamines modify the brain and alter behavior through mechanisms generally attributed to their ability to regulate extracellular dopamine concentrations. However, the actions of amphetamine are also linked to adaptations in glutamatergic signaling. We report here that when amphetamine enters dopamine neurons through the dopamine transporter, it stimulates endocytosis of an excitatory amino acid transporter, EAAT3, in dopamine neurons. Consistent with this decrease in surface EAAT3, amphetamine potentiates excitatory synaptic responses in dopamine neurons. We also show that the process of internalization is dynamin- and Rho-mediated and requires a unique sequence in the cytosolic C terminus of EAAT3. Introduction of a peptide based on this motif into dopamine neurons blocks the effects of amphetamine on EAAT3 internalization and its action on excitatory responses. These data indicate that the internalization of EAAT3 triggered by amphetamine increases glutamatergic signaling and thus contributes to the effects of amphetamine on neurotransmission.

Tolerance to repeated morphine administration is associated with increased potency of opioid agonists.

Tolerance to the pain-relieving effects of opiates limits their clinical use. Although morphine tolerance is associated with desensitization of μ-opioid receptors, the underlying cellular mechanisms are not understood. One problem with the desensitization hypothesis is that acute morphine does not readily desensitize μ-opioid receptors in many cell types. Given that neurons in the periaqueductal gray (PAG) contribute to morphine antinociception and tolerance, an understanding of desensitization in PAG neurons is particularly relevant. Opioid activity in the PAG can be monitored with activation of G-protein-mediated inwardly rectifying potassium (GIRK) currents. The present data show that opioids have a biphasic effect on GIRK currents in morphine tolerant rats. Opioid activation of GIRK currents is initially potentiated in morphine (EC50=281 nM) compared to saline (EC50=8.8 μM) pretreated rats as indicated by a leftward shift in the concentration–response curve for met-enkephalin (ME)-induced currents. These currents were inhibited by superfusion of the μ-opioid receptor antagonist β-funaltrexamine (β-FNA) suggesting that repeated morphine administration enhances agonist stimulation of μ-opioid receptor coupling to G-proteins. Although supersensitivity of μ-opioid receptors in the PAG is counterintuitive to the development of tolerance, peak GIRK currents from tolerant rats desensitized more than currents from saline pretreated rats (56% of peak current after 10 min compared to 15%, respectively). These data indicate that antinociceptive tolerance may be triggered by enhanced agonist potency resulting in increased desensitization of μ-opioid receptors.

Tolerance to the Antinociceptive Effect of Morphine in the Absence of Short-Term Presynaptic Desensitization in Rat Periaqueductal Gray Neurons

Opioids activate the descending antinociceptive pathway from the ventrolateral periaqueductal gray (vlPAG) by both pre- and postsynaptic inhibition of tonically active GABAergic neurons (i.e., disinhibition). Previous research has shown that short-term desensitization of postsynaptic μ-opioid receptors (MOPrs) in the vlPAG is increased with the development of opioid tolerance. Given that pre- and postsynaptic MOPrs are coupled to different signaling mechanisms, the present study tested the hypothesis that short-term desensitization of presynaptic MOPrs also contributes to opioid tolerance. Twice-daily injections of morphine (5 mg/kg s.c.) for 2 days caused a rightward shift in the morphine dose-response curve on the hot plate test (D50 = 9.9 mg/kg) compared with saline-pretreated (5.3 mg/kg) male Sprague-Dawley rats. In vitro whole-cell patch-clamp recordings from vlPAG slices revealed that inhibition of evoked inhibitory postsynaptic currents (eIPSCs) by the MOPr-selective agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin was decreased in morphine-tolerant (EC50 = 708 nM) compared with saline-pretreated rats (EC50 = 163 nM). However, short-term desensitization of MOPr inhibition of eIPSCs was not observed in either saline- or morphine-pretreated rats. Reducing the number of available MOPrs with the irreversible opioid receptor antagonist, β-chlornaltrexamine decreased maximal MOPr inhibition with no evidence of desensitization, indicating that the lack of observed desensitization is not caused by receptor reserve. These results demonstrate that tolerance to the antinociceptive effect of morphine is associated with a decrease in presynaptic MOPr sensitivity or coupling to effectors, but this change is independent of short-term MOPr desensitization.