Bruce D. Wyse

H-Ras Signaling and K-Ras Signaling Are Differentially Dependent on Endocytosis

ABSTRACT Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Caveolin Interacts with the Angiotensin II Type 1 Receptor during Exocytic Transport but Not at the Plasma Membrane

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R·caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.

The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

BackgroundThe vanilloid receptor 1 (TRPV1) is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA) pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described.ResultsIn the present studies we investigated the hypothesis that the μ opioid receptor (MOP) agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+) responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine.Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1.ConclusionThe results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

Small Molecule Angiotensin II Type 2 Receptor (AT2R) Antagonists as Novel Analgesics for Neuropathic Pain: Comparative Pharmacokinetics, Radioligand Binding, and Efficacy in Rats

OBJECTIVE Neuropathic pain is an area of unmet clinical need. The objective of this study was to define the pharmacokinetics, oral bioavailability, and efficacy in rats of small molecule antagonists of the angiotensin II type 2 receptor (AT₂R) for the relief of neuropathic pain. DESIGN AND METHODS Adult male Sprague-Dawley (SD) rats received single intravenous (1-10 mg/kg) or oral (5-10 mg/kg) bolus doses of EMA200, EMA300, EMA400 or EMA401 (S-enantiomer of EMA400). Blood samples were collected immediately pre-dose and at specified times over a 12- to 24-hour post-dosing period. Liquid chromatography tandem mass spectrometry was used to measure plasma drug concentrations. Efficacy was assessed in adult male SD rats with a unilateral chronic constriction injury (CCI) of the sciatic nerve. RESULTS After intravenous administration in rats, mean (±standard error of the mean) plasma clearance for EMA200, EMA300, EMA400, and EMA401 was 9.3, 6.1, 0.7, and 1.1 L/hour/kg, respectively. After oral dosing, the dose-normalized systemic exposures of EMA400 and EMA401 were 20- to 30-fold and 50- to 60-fold higher than that for EMA300 and EMA200, respectively. The oral bioavailability of EMA400 and EMA401 was similar at ∼30%, whereas it was only 5.9% and 7.1% for EMA200 and EMA300, respectively. In CCI rats, single intraperitoneal bolus doses of EMA200, EMA300, and EMA400 evoked dose-dependent pain relief. The pain relief potency rank order in CCI rats was EMA400 > EMA300 > EMA200 in agreement with the dose-normalized systemic exposure rank order in SD rats. CONCLUSION The small molecule AT₂R antagonist, EMA401, is in clinical development as a novel analgesic for the relief of neuropathic pain.

Synthesis and biological evaluation of an orally active glycosylated endomorphin-1

The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability and membrane permeability of 2 while retaining μ-opioid receptor binding affinity and agonist activity. Analogue 2 produced dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of neuropathic pain with an ED(50) of 8.3 (± 0.8) μmol/kg. The corresponding ED(50) for morphine was 2.6 (± 1.4) μmol/kg. Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED(50) = 19.6 (± 1.2) μmol/kg), which was comparable with that of morphine (ED(50) = 20.7 (±3.6) μmol/kg). Antineuropathic effects of analogue 2 were significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.

A small molecule angiotensin II type 2 receptor (AT2R) antagonist produces analgesia in a rat model of neuropathic pain by inhibition of p38 Mitogen-Activated Protein Kinase (MAPK) and p44/p42 MAPK activation in the dorsal root ganglia

OBJECTIVE There is an unmet clinical need for novel analgesics for neuropathic pain. This study was designed to elucidate the mechanism through which EMA300, a small molecule antagonist of the angiotensin II type 2 receptor (AT₂R) with >1,000-fold selectivity over the angiotensin II type 1 receptor, produces analgesia in a rodent model of neuropathic pain. DESIGN AND METHODS Groups of AT₂R knockout, hemizygotes, and wild-type mice with a chronic constriction injury (CCI) of the sciatic nerve received single intraperitoneal (i.p.) bolus doses of EMA300 (100 or 300 mg/kg), and analgesic efficacy was assessed. Groups of control, sham-operated, and CCI rats were euthanized and perfusion fixed. Lumbar dorsal root ganglia (DRGs) were removed for investigation of the mechanism through which EMA300 alleviates neuropathic pain. RESULTS EMA300 analgesia was abolished in AT₂R knockout CCI mice with intermediate responses in the hemizygotes, affirming the AT₂R as the target mediating EMA300 analgesia. In CCI rats, DRG immunofluorescence (IF) levels for angiotensin II, the main endogenous ligand of the AT₂R, were increased ∼1.5-2.0-fold (P < 0.05) cf. sham-controls. Mean DRG IF levels for activated p38 (pp38) and activated p44/p42 (pp44/pp42) MAPK were also increased ∼1.5-2.0-fold (P < 0.05) cf. sham-controls. At the time of peak EMA300 analgesia in CCI rats, mean DRG levels of pp38 MAPK and pp44/pp42 MAPK (but not angiotensin II) were reduced to match the respective levels in sham-controls. CONCLUSION Augmented angiotensin II/AT₂R signaling in the DRGs of CCI rats is attenuated by EMA300 to block p38 MAPK and p44/p42 MAPK activation, a mechanism with clinical validity for alleviating neuropathic pain.

Lipo-endomorphin-1 derivatives with systemic activity against neuropathic pain without producing constipation.

To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (1 = Tyr-Pro-Trp-Phe-NH2), the N-terminus of the peptide was modified with 2-aminodecanoic acid, resulting in compound 3. Tyr in compound 1 was replaced with 2,6-dimethyltyrosine yielding compound 2. Derivative 2 was also substituted with 2-aminodecanoic acid producing compound, 4. Lipoamino acid-modified derivatives showed improved metabolic stability and membrane permeability while maintaining high μ-opioid (MOP) receptor binding affinity and acting as a potent agonist. In vivo studies showed dose-dependent antinociceptive activity following intravenous (i.v.) administration of compounds 3 and 4 in a chronic constriction injury (CCI)-rat model of neuropathic pain with ED50 values of 1.22 (±0.93) and 0.99 (±0.89) µmol/kg, respectively. Pre-treatment of animals with naloxone hydrochloride significantly attenuated the anti-neuropathic effects of compound 3, confirming the key role of opioid receptors in mediating antinociception. In contrast to morphine, no significant constipation was produced following i.v. administration of compound 3 at 16 µmol/kg. Furthermore, following chronic administration of equi-potent doses of compound 3 and morphine to rats, there was less antinociceptive tolerance for compound 3 compared with morphine.

Novel perspectives on pituitary and brain angiotensinogen.

All the angiotensin peptides originate from angiotensinogen, a glycoprotein synthesized by several tissues, including the brain and the anterior pituitary. In the rat, immunohistochemistry has been used to localize angiotensinogen in gonadotropes and in uncharacterized cells surrounding sinusoids. Both cell types are capable of secreting angiotensinogen in cell culture; only the gonadotropes contain angiotensin II (AngII) and are capable of secreting it in culture. It has been asserted that the perisinusoidal cells are the only source of angiotensinogen for the generation of AngII by gonadotropes. Our current data favor the existence of a complete intracellular renin-angiotensin system (RAS) in gonadotropes and a separate extracellular system which utilizes the high concentration of angiotensinogen from perisinusoidal cells. Furthermore, we postulate that gonadotrope AngII serves mainly reproductive functions, while the proximity of angiotensinogen-secreting cells to folliculostellate cells, and their access to the intercellular sinusoidal and follicular spaces, places the extracellular RAS in a strategic position to affect pituitary growth and the mediation of acute-phase immune responses. In the rat brain, angiotensinogen is expressed by the 16-18th day of fetal life and by areas generally concerned with vasopressor, electrolyte, and fluid homeostasis. Antisense deoxyoligonucleotides to angiotensinogen mRNA lower blood pressure in hypertensive rats and inhibit in vitro growth of neuroblastoma cells, indicating a significant role for angiotensinogen in mitogenic and homeostatic functions. It is commonly agreed that astrocytes express angiotensinogen. Neuronal angiotensinogen has also been demonstrated by immunohistochemistry, as a secretion from neuronal cell cultures, and by reverse-transcriptase polymerase chain reaction. The fate of secreted astrocytic and neuronal angiotensinogen remains obscure. Angiotensinogen is regulated in a tissue-specific manner with smaller or absent responses observed for brain tissue. By using astrocyte and neuronal cultures the actions on angiotensinogen production of growth hormone, IGF-1, inflammatory lipopolysaccharide, and phorbol ester have been examined. Recent observations show that angiotensinogen is regulated positively or negatively by glucocorticoids and that a positive synergism between cAMP and glucocorticoids exists. On the basis of analogous systems for other proteins, a scheme involving glucocorticoid receptors, CREB, and AP-1 transcription factors is formulated to explain glucocorticoid-cAMP interactions. These transcriptional interactions may form a significant functional link between the RAS and adrenergic mechanisms.

SEX DIFFERENCES IN THE PHARMACOKINETICS, OXIDATIVE METABOLISM AND ORAL BIOAVAILABILITY OF OXYCODONE IN THE SPRAGUE-DAWLEY RAT

1 The pharmacokinetics and oxidative metabolism of oxycodone were investigated following intravenous and oral administration in male and female Sprague‐Dawley (SD) rats. 2 High‐performance liquid chromatography (HPLC)–electrospray ionization (ESI)–tandem mass spectrometry (MS‐MS) was used to quantify plasma concentrations of oxycodone and its oxidative metabolites noroxycodone and oxymorphone following administration of single bolus intravenous (5 mg/kg) and oral (10 mg/kg) doses of oxycodone. 3 The mean (±SEM) clearance of intravenous oxycodone was significantly higher in male than female SD rats (4.9 ± 0.3 vs 3.1 ± 0.3 L/h per kg, respectively; P < 0.01). Mean areas under the plasma concentration versus time curves (AUC) for oxycodone were significantly higher in female than male SD rats following intravenous (~1.6‐fold; P < 0.01) and oral (~sevenfold; P < 0.005) administration. 4 The oral bioavailability of oxycodone was low (at 1.2 and 5.0%, respectively) in male and female SD rats, a finding consistent with high first‐pass metabolism. Noroxycodone : oxycodone AUC ratios were significantly higher in male than female SD rats after intravenous (~2.4‐fold; P < 0.005) and oral (~12‐fold; P < 0.005) administration. 5 Circulating oxymorphone concentrations remained very low following both routes of administration. Noroxycodone : oxymorphone AUC ratios were greater in male than female SD rats after intravenous (~13‐ and fivefold, respectively) and oral (~90‐ and sixfold, respectively) administration. 6 Sex differences were apparent in the pharmacokinetics, oxidative metabolism and oral bioavailability of oxycodone. Systemic exposure to oxycodone was greater in female compared with male SD rats, whereas systemic exposure to metabolically derived noroxycodone was higher in male than female SD rats. 7 Oral administration of oxycodone to the SD rat is a poor model of the human for the study of the pharmacodynamic effects of oxycodone.

In vivo profiling of seven common opioids for antinociception, constipation and respiratory depression: no two opioids have the same profile

For patients experiencing inadequate analgesia and intolerable opioid‐related side effects on one strong opioid analgesic, pain relief with acceptable tolerability is often achieved by rotation to a second strong opioid. These observations suggest subtle pharmacodynamic differences between opioids in vivo. This study in rats was designed to assess differences between opioids in their in vivo profiles.