Agonist-induced Sensitization Research Articles

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.

Regulation of the crossbridge cycle in vascular smooth muscle by cAMP signalling

Urocortin, a novel vasodilatory peptide related to the corticotropin-releasing factor (CRF) increased cAMP levels to 220.8 +/- 27.6% of control in rat tail arteries. The effect was completely abolished by the adenylyl cyclase inhibitor, SQ22536 (100 microM). Urocortin also decreased phosphorylation of the regulatory light chains of myosin (MLC20) in rat tail arteries stimulated with high K+ from 27.5 +/- 0.9% (control) to 13 +/- 2% (n = 5). This suggests that urocortin relaxes blood vessels via cAMP-mediated dephosphorylation of MLC20. Previously we have shown that urocortin-induced vasodilation can be ascribed to a decrease in Ca2+ -sensitivity of tension and activation of smooth muscle myosin phosphatase (SMPP-1M). In this study, we provide evidence that urocortin-induced Ca2+ -desensitization does not affect agonist-induced Ca2+ -sensitization. Urocortin relaxed alpha-toxin permeabilized mouse tail arteries preconstricted with pCa 6.1, but did not prevent the Ca2+ -sensitization induced by 10 microM 5-HT, 100 microM norepinephrine (NE) or 1 microM GTPgammaS. In keeping, the maximally relaxing concentration of urocortin (100 nM) had no effect on the concentration dependence of the phenylephrine-induced Ca2+ -sensitization. By contrast, treatment with the cAMP analogue, cBIMPS (100 microM), or the Rho kinase inhibitor, H-1152 (3 microM) relaxed the mouse vessels to a greater extend and completely inhibited phenylephrine (PE) induced sensitization. The lack of effect of urocortin on agonist-induced sensitization could be due to a alpha-adrenergic receptor mediated inhibition of cAMP generation. Furthermore PE induced Ca2+ -sensitization was reported to occur independent of changes in MLC20 phosphorylation involving caldesmon. Our results are compatible with a model in which urocortin/cAMP signalling only affects the myosin linked regulation of vascular tone while cBIMPS may inactivate in addition the MLC20 phosphorylation independent pathway.

Using molecular tools to dissect the role of Galphas in sensitization of AC1.

Short-term activation of Galpha(i/o)-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of Galpha(i/o)-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity after subsequent activation by Galpha(s) or forskolin. Several indirect observations have suggested the involvement of increased Galpha(s)-adenylyl cyclase interactions in the expression of sensitization; however, evidence supporting a direct role for Galpha(s) has not been well established. In the present report, we used two genetic approaches to further examine the role of Galpha(s) in heterologous sensitization of Ca(2+)-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed Galpha(s)-insensitive mutants of AC1 (F293L and Y973S) that retained sensitivity to Ca2+ and forskolin activation. Persistent (2 h) activation of the D2 dopamine receptor resulted in a significant augmentation of basal or Ca(2+)- and forskolin-stimulated AC1 activity; however, sensitization of Galpha(s)-insensitive mutants of AC1 was markedly reduced compared with wild-type AC1. In the second strategy, we examined the requirement of an intact receptor-Galpha(s) signaling pathway for the expression of sensitization using dominant-negative Galpha(s) mutants (alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S) to disrupt D1 dopamine receptor activation of recombinant AC1. D1 dopamine receptor-Galpha(s) signaling was attenuated in the presence of alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S, but D2 agonist-induced sensitization of Ca(2+)-stimulated AC1 activity was not altered. Together, the present findings directly support the hypothesis that the expression of sensitization of AC1 involves Galpha(s)-adenylyl cyclase interactions.

Adrenergic agonists induce heterologous sensitization of adenylate cyclase in NS20Y-D 2L cells

Adenylate cyclase activity in NS20Y cells expressing D 2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D 2 receptors. Furthermore, chronic (18 h) activation of D 2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D 2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Gα s or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.

Effects of the NMDA receptor antagonist, D-CPPene, on sensitization to the operant decrement produced by naloxone in morphine-treated rats.

Sensitization to the rate-decreasing effects of opioid antagonists induced by acute pretreatment with opioid agonists has been suggested to reflect initial changes in opioid systems that underlie physical dependence. Glutamate receptors are implicated in the development and expression of opioid dependence, and antagonists acting at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors have been shown repeatedly to attenuate the severity of opioid withdrawal. The present study evaluated the ability of a competitive NMDA receptor antagonist, D-CPPene (SDZ EAA 494; 3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid), to affect morphine-induced sensitization to naloxone in rats trained to lever-press on a multiple-trial, fixed-ratio 10 schedule of food reinforcement. D-CPPene (0.3-3 mg/kg) was administered either 4 h or 30 min prior to the test session. Morphine (10 mg/kg) or its vehicle was administered 4 h before naloxone challenge (0.3-3 mg/kg). D-CPPene failed to prevent morphine-induced potentiation of the naloxone-produced decrement in operant performance. Thus, these results suggest that agonist-induced sensitization to behavioral effects of opioid antagonists may be insensitive to NMDA receptor blockade.

Ontogeny of G-protein expression: control by β-adrenoceptors

Cardiac cell homeostasis is maintained in the face of excessive β-adrenoceptor stimulation through the process of desensitization. Desensitization is not an inherent property of these cells but rather is acquired during development; neonates given β-agonists actually show heterologous sensitization, involving changes in the expression and catalytic activity of adenylyl cyclase (AC) as well as an increased receptor/G-protein coupling. The current study examines the role of specific G-protein components, G sα and G iα, in the ontogeny of β-adrenoceptor responses and in the transition from agonist-induced sensitization to desensitization. Between postnatal days (PN) 6 and 15 there was a significant decrease in the 52 kDa isoform of G sα with no accompanying change of the 45 kDa form; over the same period, G iα3 also declined substantially. In contrast, the 45 kDa isoform of G sα and G iα1,2 remained fairly constant over the same period and fluoride-stimulated AC activity increased. Treatment with isoproterenol on PN2–5 did not result in any significant changes in G sα expression but robustly decreased G iα1,2. These changes were accompanied by heterologous sensitization of AC activity at the level of AC itself, evidenced by equivalent increases in the enzymatic response to fluoride and forskolin–Mn 2+. Isoproterenol given to older animals (PN11–14) also caused specific loss of G i protein, in this case targeting G iα3, whereas G sα again was unchanged; in contrast to the younger group, the older animals displayed heterologous desensitization of AC at the level of G-protein function (specific loss of the fluoride response). These results indicate that the normal ontogenetic increase of cardiac β-adrenoceptor coupling to AC is not dependent on the absolute amount of G-proteins, nor on the relative balance of stimulatory (G s) and inhibitory (G i) subunits. However, the ability of receptor stimulation to downregulate G iα1,2, an event which is specific to immature cardiac cells, is likely to be an important component of the resistance of the fetal/neonatal heart to agonist-induced desensitization and hypertrophy. The maintenance of cardiac β-adrenoceptor signaling in the face of intense stimulation is likely to play an important role in the physiologic adaptations necessary to the perinatal transition.

Short- and long-term heterologous sensitization of adenylate cyclase by D4 dopamine receptors.

The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.

Increased sensitization of the myofilaments in rat neonatal portal vein: a potential mechanism.

The contractile regulation of neonatal smooth muscle was studied in rat neonatal portal vein. Strips of beta-escin-permeabilized portal vein from 3- to 5-day-old rats and 5- to 6-week-old rats were mounted on a 'bubble chamber' in calcium solutions buffered with 10 mM EGTA. Although the overall tension development was lower in neonatal muscle as expected, the calcium sensitivity of the neonatal permeabilized portal vein was significantly higher than in developed portal vein. Endothelin-1-induced sensitization of the myofilaments was investigated. Endothelin-1 produced an increase in tension of permeabilized neonatal portal vein in a calcium solution buffered with 10 mM EGTA. This sensitization was proportionally higher in neonatal than in developed smooth muscle, despite similar initial submaximal calcium contractions. GTP gamma S-induced calcium sensitization was also proportionally higher in neonatal permeabilized strips than in fully developed smooth muscle. These changes may be due to alterations in the intracellular signalling pathways which mediate calcium sensitization in smooth muscle. As some endothelin-1-mediated responses are known to occur via activation of the heterotrimeric GTP-binding protein, Gq, the levels of protein expression of Gq alpha were studied. In membrane preparations from neonatal rat portal vein the expression of Gq alpha was significantly higher than in portal vein membrane preparations loaded with equal protein from 5- to 6-week-old rats. In conclusion, agonist-induced sensitization of the myofilaments was higher in neonatal rat portal vein than in fully developed portal vein. This difference is the result of changes in intracellular signalling and may be partly produced by the greater expression of Gq alpha observed in neonatal portal vein.

The effect of nigral implantation on sensitization to dopamine agonists in 6-hydroxydopamine-lesioned rats

The implantation of fetal nigral tissue into the striatum of patients with Parkinson's disease is a promising approach to treatment which may produce clinical benefit partly by influencing drug responsiveness. The purpose of the present study was to determine the pharmacological mechanisms which drug response changes by measuring to what extent sensitization produced by repeated apomorphine treatment was attenuated by tissue implantation in rats with nigrostriatal lesions. Prior to implantation of nigral cell suspensions, the daily administration of apomorphine to rats with unilateral 6-hydroxydopamine lesions produced a progressive increase in the magnitude and duration of rotational behaviour. After implantation, apomorphine-induced rotational effects were reduced to levels observed upon the initial exposure to drug and did not increase following repeated treatment. Attenuated responses to selective D 1 and D 2 agonists were also observed after implantation. In vehicle-implanted rats, the initial response to apomorphine was attenuated but then increased following repeated apomorphine administration. No attenuation in responses to selective D 1 and D 2 agonists was observed in this group. Cell suspensions prepared from fresh and cyropreserved tissue produced similar behavioural effects, even though the volume of transplanted striatum exhibiting tyrosine hydroxylase activity was greater with fresh tissue. The duration of rotational behaviour induced by apomorphine was not affected by cell implantation. These findings suggest that the expression of sensitization in an animal model of parkinsonism may disappear after a period without drug treatment. Implantation of nigral tissue may produce beneficial results in parkinsonism by limiting the development of dopamine agonist-induced sensitization.

Ontogeny of dopamine agonist-induced sensitization: role of NMDA receptors.

In contrast to adults, preweanling rats exhibit behavioral sensitization for only a few days after cessation of dopamine (DA) agonist treatment. The reasons for this ontogenetic difference are uncertain, but maturational changes in the N-methyl-D-aspartate (NMDA) receptor may be responsible, since stimulation of these receptors is necessary for the development of DA agonist-induced sensitization in adult rats. The purpose of the present study was to examine the relationship between NMDA receptor functioning and DA agonist-induced sensitization during the preweanling period. To that end, 17-day-old rats were injected (i.p.) on 4 consecutive days with saline or 0.3 mg/kg dizocilpine (a non-competitive NMDA receptor antagonist) followed, 30 min later, by an injection of saline, 2.5 mg/kg amphetamine (an indirect DA agonist), or 1.0 mg/kg NPA (a direct DA agonist). Sensitization was tested 2 days later (i.e., at 22 days of age), with rats receiving a challenge injection of saline, amphetamine, NPA, or dizocilpine. Results showed that the NMDA antagonist had adult-like effects on the behavioral sensitization of preweanling rats, as amphetamine- and NPA-induced sensitization were eliminated by dizocilpine pretreatment. When given alone, dizocilpine substantially increased the locomotor activity (i.e., line-crosses) of preweanling rats, an effect that became sensitized with repeated drug treatment. Lastly, preweanling rats already sensitized to dizocilpine did not exhibit cross-sensitization to amphetamine or NPA. Thus, with few exceptions, NMDA receptor stimulation appears to modulate sensitization in a similar fashion across ontogeny. This finding suggests that maturational differences in the NMDA receptor system are not responsible for the lack of long-term sensitization in the younger animal.

Naloxone pretreatment blocks acute morphine-induced sensitization to naltrexone.

The present experiment was designed to examine whether the acute sensitization to naltrexone that is induced by a single dose of morphine could be blocked by pretreatment with naloxone. Food-deprived male Sprague-Dawley rats were trained to respond for food on a multiple-trial fixed interval 3-min schedule. Reinforcement was contingent upon a response within a 10-s limited hold period following a fixed interval of 3 min. A trial consisted of three fixed intervals separated by a 10-min timeout period during which responses were not reinforced. The rate decreasing effects of the opioid antagonist naltrexone were determined by cumulative dosing. Pretreatment with morphine (3.0 mg/kg, SC) resulted in a 70-fold increase in sensitivity to the response rate decreasing effect of naltrexone compared with saline pretreatment. The increased sensitivity was dose-dependently blocked by naloxone administration 10 min before morphine. The blockade by naloxone was overcome by increasing the pretreatment dose of morphine to 10 mg/kg. The results provide further evidence that acute agonist-induced sensitization to the rate-reducing effects of naltrexone is mediated by opioid receptors.

Latent sensitization to apomorphine following repeated low doses.

In two experiments, the effect of repeated injections of apomorphine on locomotor activity of rats was determined. In each experiment, different groups of rats were injected with either apomorphine (0.2, 1.0, or 5.0 mg/kg) or vehicle at either 24 or 72 hr intervals and tested for locomotor activity in photocell arenas. In Experiment 2, following 13 treatment sessions with various doses, all groups were first tested for activity following a 5.0 mg/kg dose of apomorphine and then given vehicle only prior to the final activity test session. Major findings were as follows: (a) repeated injections of 1.0 and 5.0 mg/kg apomorphine produced a progressively greater increase in activity with each injection (i.e., sensitization); (b) injections of 0.2 mg/kg of apomorphine produced a slight inhibition of activity, which did not change with repeated injections; (c) prior treatment with 0.2 mg/kg of apomorphine resulted in a significantly greater activity increase following a 5.0 mg/kg dose of apomorphine than did prior vehicle treatments; and (d) chronic pretreatment of rats with apomorphine did not affect their activity level following a vehicle injection. These findings suggest that sensitization to apomorphine is a graded, rather than an all or none, phenomenon dependent on the dose of apomorphine repeatedly administered. In addition, these results are inconsistent with autoreceptor tolerance and conditioning explanations of dopamine agonist-induced sensitization effects.