Gracious R. Ross

Blockade of Endocannabinoid Hydrolytic Enzymes Attenuates Precipitated Opioid Withdrawal Symptoms in Mice

Δ9-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB1 and CB2 cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB1 receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB1 receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.

Morphine tolerance in the mouse ileum and colon

Repeated administration of morphine is associated with tolerance to its antinociceptive properties. However, constipation remains the major side effect of chronic exposure to morphine. In contrast, previous studies suggest that tolerance to opioids develops in the ileum of several species. In this study, we provide evidence that constipation may arise due to a lack of tolerance development to morphine in the colon. Mice received implants with either placebo or 75 mg of morphine pellets, and they were examined for morphine tolerance to antinociception, defecation, and intestinal and colonic transit after 72 h. Tissues were obtained from the ileum and distal colon, and contractile responses were measured from longitudinal and circular muscle preparations. In morphine-pelleted mice, a 5.5-fold tolerance developed to antinociception after 72 h, and a 53.2-fold tolerance developed in mice that received an additional daily morphine injection. In both models, intestinal transit but not defecation or colonic transit developed tolerance. In isolated longitudinal muscles, electrical field stimulation-induced cholinergic contractions were dose-dependently inhibited by morphine in both the ileum and colon of placebo pelleted with a pD2 of 7.1 ± 0.4 and 7.8 ± 0.4, respectively. However, the dose response to morphine inhibition was shifted to the right for the ileum from morphine-pelleted mice (pD2 = 5.1 ± 0.4) but not the colon (pD2 = 6.9 ± 0.4). In circular muscle preparations, morphine induced atropine-insensitive contractions in both tissue segments. Tolerance to morphine developed in the ileum but not the colon upon repeated administration of morphine. These findings indicate that a lack of tolerance development in the colon is the basis for opioid bowel dysfunction.

The role of β-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract

β-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of β-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 μM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC50 values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from β-arrestin2 knockout mice. β-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of β-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 μM) exposure. Naloxone prevented the decrease in β-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in β-arrestin2 occurred. We conclude that a decrease in β-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Nitrotyrosylation of Ca2+ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis

Basal levels of c-Src kinase are known to regulate smooth muscle Ca2+ channels. Colonic inflammation results in attenuated Ca2+ currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase in experimental colitis. Ca2+-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K+. The Emax to CaCl2 was significantly less in inflamed tissues (38.4 ± 7.6%) than controls, indicative of reduced Ca2+ influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a selective Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD2 from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/peroxynitrite combination, the maximal contraction to CaCl2 was reduced by 46 ± 7% in controls but unaffected in inflamed tissues (13 ± 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca2+ currents by 84.1 ± 3.9% in normal but only 36.2 ± 13% in inflamed tissues. Neither the Ca2+ channel Cav1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca2+ channel protein expression but increased nitrotyrosylated-Ca2+ channel proteins during inflammation. These data suggest that post-translational modification of Ca2+ channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca2+ influx.

Endothelium-Independent Relaxation by Adrenomedullin in Pregnant Rat Mesenteric Artery: Role of cAMP-Dependent Protein Kinase A and Calcium-Activated Potassium Channels

The mechanisms of relaxation of adrenomedullin were investigated in isolated mesenteric artery from pregnant rats. Adrenomedullin (1 nM–0.3 μM) produced concentration-dependent relaxation of endothelium-denuded mesenteric artery rings precontracted with norepinephrine at a concentration required to produce 70% of maximal response (ED70). The concentration-response curve of adrenomedullin was shifted to the right by adrenomedullin receptor antagonist adrenomedullin22–52 (10 μM) or calcitonin gene-related peptide8–37 (1 μM). Inhibition of adenylate cyclase by 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) (10 μM) or protein kinase A [Rp-cyclic adenosine monophosphorothioate (Rp-cAMP); 10 μM] reduced the adrenomedullin-induced relaxation to the same magnitude. Adrenomedullin increased the intracellular cAMP level from 0.38 ± 0.07 to 2.00 ± 0.47 pmol/mg tissues, which was completely inhibited by adrenomedullin22–52 (100 μM). Extracellular high potassium (80 mM), which inactivates the potassium channels, reduced the adrenomedullin-induced relaxation. Blockade of ATP-sensitive, voltage-gated, or inward rectifier potassium channels did not affect the adrenomedullin-induced relaxation. Blockade of calcium-activated K+ channels (KCa) by tetraethylammonium (1 mM) or iberiotoxin (100 nM) inhibited the adrenomedullin-induced relaxation, whereas there was no additional inhibition by SQ22536 or Rp-cAMP when KCa channels were already inhibited. In conclusion, this study provides evidence that cAMP-dependent protein kinase A and KCa channels seem to mediate as the cellular pathways in the adrenomedullin-induced endothelium-independent relaxation of mesenteric artery from pregnant rats.

Adrenomedullin Relaxes Rat Uterine Artery: Mechanisms and Influence of Pregnancy and Estradiol

Uterine arteries play a major role in regulating uteroplacental blood flow. Failure to maintain blood flow to the uteroplacental compartment during pregnancy often results in intrauterine growth retardation. Immunohistochemical staining of adrenomedullin (AM), an endogenous vasoactive peptide, in uterine artery was intense in pregnant compared to nonpregnant rats, but it is not known whether AM directly relaxes uterine artery or not. In this study, we elucidated the mechanisms of uterine artery relaxation by AM and its regulation by pregnancy and female sex steroids. AM was able to relax uterine artery, and this relaxation was influenced positively by pregnancy and estradiol as evidenced by the increased pD(2) and E(max) values of AM. Both pregnancy and estradiol treatment to ovariectomized rats amplified RAMP(3) expression in uterine arteries while progesterone had no effect. AM-induced uterine artery relaxation is predominantly endothelium-dependent. The AM receptor antagonist CGRP(8-37) is more potent than AM(22-52) in inhibiting the AM relaxation, indicating the involvement of AM(2) receptor subtype. Moreover, AM uses the classical nitric oxide-cyclic guanosine monophosphate pathway along with K(Ca) channels to mediate the vasodilatory effect in uterine artery. In conclusion, sensitivity of uterine artery to AM-induced relaxation is increased with pregnancy or estradiol treatment by increasing RAMP(3) expression, suggesting an important role for AM in regulating the uterine hemodynamics, probably maintaining uterine blood flow during pregnancy and in pre- and postmenopausal cardiovascular adaptation differences.

Evidence for the putative cannabinoid receptor (GPR55)-mediated inhibitory effects on intestinal contractility in mice.

Background: Cannabinoids inhibit intestinal motility via presynaptic cannabinoid receptor type I (CB1) in enteric neurons while cannabinoid receptor type II (CB2) receptors are located mainly in immune cells. The recently de-orphanized G-protein-coupled receptor, GPR55, has been proposed to be the ‘third’ cannabinoid receptor. Although gene expression of GPR55 is evident in the gut, functional evidence for GPR55 in the gut is unknown. In this study, we tested the hypothesis that GPR55 activation inhibits neurogenic contractions in the gut. Methods: We assessed the inhibitory effect of the atypical cannabinoid O-1602, a GPR55 agonist, in mouse colon. Isometric tension recordings in colonic tissue strips were used from either wild-type, GPR55–/– or CB1–/–/CB2–/– knockout mice. Results: O-1602 inhibited the electrical field- induced contractions in the colon strips from wild-type and CB1–/–/CB2–/– in a concentration-dependent manner, suggesting a non-CB1/CB2 receptor-mediated prejunctional effect. The concentration-dependent response of O-1602 was significantly inhibited in GPR55–/– mice. O-1602 did not relax colonic strips precontracted with high K+ (80 mmol/l), indicating no involvement of Ca2+ channel blockade in O-1602-induced relaxation. However, 10 µmol/l O-1602 partially inhibited the exogenous acetylcholine (10 µmol/l)-induced contractions. Moreover, we also assessed the inhibitory effects of JWH015, a CB2/GPR55 agonist on neurogenic contractions of mouse ileum. Surprisingly, the effects of JWH015 were independent of the known cannabinoid receptors. Conclusion: Taken together, these findings suggest that activation of GPR55 leads to inhibition of neurogenic contractions in the gut and are predominantly prejunctional.

The effect of tyrosine nitration of L-type Ca2+ channels on excitation-transcription coupling in colonic inflammation.

Background and purpose:  Excitation–transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L‐type Ca2+ channels induces phosphorylation of the transcription factor, cyclic‐AMP response element binding protein (CREB) and downstream activation of the cyclic‐AMP response element (CRE) promoter regions. Tyrosine nitration of Ca2+ channels attenuates interactions with c‐Src kinase, decreasing Ca2+ channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca2+ channel mediated phosphorylation of CREB and CRE activation.

Opioid-induced hypernociception is associated with hyperexcitability and altered tetrodotoxin-resistant Na channel function of dorsal root ganglia

Opiates are potent analgesics for moderate to severe pain. Paradoxically, patients under chronic opiates have reported hypernociception, the mechanisms of which are unknown. Using standard patch-clamp technique, we examined the excitability, biophysical properties of tetrodotoxin-resistant (TTX-R) Na(+) and transient receptor potential vanilloid 1 (TRPV1) channels of dorsal root ganglia neurons (DRG) (L(5)-S(1)) from mice pelleted with morphine (75 mg) or placebo (7 days). Hypernociception was confirmed by acetic acid-writhing test following 7-day morphine. Chronic morphine enhanced the neuronal excitability, since the rheobase for action potential (AP) firing was significantly (P < 0.01) lower (38 ± 7 vs. 100 ± 15 pA) while the number of APs at 2× rheobase was higher (4.4 ± 0.8 vs. 2 ± 0.5) than placebo (n = 13-20). The potential of half-maximum activation (V(1/2)) of TTX-R Na(+) currents was shifted to more hyperpolarized potential in the chronic morphine group (-37 ± 1 mV) vs. placebo (-28 ± 1 mV) without altering the V(1/2) of inactivation (-41 ± 1 vs. -33 ± 1 mV) (n = 8-11). Recovery rate from inactivation of TTX-R Na(+) channels or the mRNA level of any Na(+) channel subtypes did not change after chronic morphine. Also, chronic morphine significantly (P < 0.05) enhanced the magnitude of TRPV1 currents (-64 ± 11 pA/pF) vs. placebo (-18 ± 6 pA/pF). The increased excitability of sensory neurons by chronic morphine may be due to the shift in the voltage threshold of activation of TTX-R Na(+) currents. Enhanced TRPV1 currents may have a complementary effect, with TTX-R Na(+) currents on opiate-induced hyperexcitability of sensory neurons causing hypernociception. In conclusion, chronic morphine-induced hypernociception is associated with hyperexcitability and functional remodeling of TTX-R Na(+) and TRPV1 channels of sensory neurons.

Vascular Hyperresponsiveness to Adrenomedullin During Pregnancy Is Associated with Increased Generation of Cyclic Nucleotides in Rat Mesenteric Artery

Abstract Cardiovascular adaptation is a hallmark of pregnancy. Here we report on vascular hyperresponsiveness to an endogenous vasodilator, adrenomedullin (ADM), during pregnancy. Intravenous administration of ADM dose dependently decreased the mean arterial pressure, and the decrease was significantly greater in pregnant compared with nonpregnant rats without affecting the heart rate. In endothelium-intact mesenteric artery precontracted by ED70 concentration of norepinephrine, the potency and efficacy of ADM in causing the vasodilation of mesenteric arterial rings from pregnant rats are significantly higher compared with nonpregnant females at diestrus. The magnitude of inhibition of concentration-dependent response to ADM by the inhibition of either soluble guanylate cyclase or adenylate cyclase was greater in pregnant rats. Moreover, ADM-induced cyclic nucleotide generation, both cGMP and cAMP, in the mesenteric artery was elevated during pregnancy and was sensitive to the receptor antagonist, ADM22–52. These findings suggest that during pregnancy the vasodilatory effects of ADM are greater and are associated with increased generation of cyclic nucleotides in resistance vessels, and these changes may be part of the cardiovascular adaptations that occur during pregnancy.