Greater Desensitization Research Articles

Allosteric Ligands of Metabotropic Glutamate Receptor 5 Have Biased Agonism and Cooperativity

Metabotropic glutamate receptor subtype 5 (mGlu5) is a G protein-coupled receptor that has emerged as an exciting new therapeutic target for schizophrenia and depression. Drugs targeting mGlu5, particularly allosteric modulators, have promising preclinical profiles; however, may also be associated with adverse effects. We hypothesise that potential on-target adverse effects of certain allosteric modulators are related to unappreciated stimulus-bias profiles. We investigated whether allosteric modulators from diverse scaffolds show differential modulation of distinct mGlu5 signalling pathways and receptor regulation processes. Allosteric modulator pharmacology of ten chemical series was assessed following both acute and prolonged exposure to mGlu5. Signalling endpoints included intracellular Ca2+ mobilization (iCa2+), inositol phosphate accumulation (IP), kinase phosphorylation and cell impedance. VU0424465, a full allosteric agonist for both iCa2+ and IP, exhibited positive cooperativity with glutamate in mediating iCa2+, but was neutral with respect to IP. Conversely, VU0360172 lacked intrinsic efficacy for iCa2+, was a partial agonist for IP, had positive cooperativity with glutamate in iCa2+ and was neutral for IP. Prolonged mGlu5 exposure to VU0424465 resulted in robust desensitization of iCa2+, whereas VU0360172 caused a moderate degree of desensitization. In contrast, greater desensitization was evident with VU0360172 compared to VU0424465 for IP. These studies will help to elucidate mechanisms of on-target mGlu5 modulator adverse effects. Further, establishing a “biased modulation fingerprint” can provide a framework for future novel biased modulator discovery.

β-Arrestin2 Regulates Cannabinoid CB1 Receptor Signaling and Adaptation in a Central Nervous System Region–Dependent Manner

Cannabinoid CB(1) receptors (CB(1)Rs) mediate the effects of ▵(9)-tetrahydrocannabinol (THC), the psychoactive component in marijuana. Repeated THC administration produces tolerance and dependence, which limit therapeutic development. Moreover, THC produces motor and psychoactive side effects. β-arrestin2 mediates receptor desensitization, internalization, and signaling, but its role in these CB(1)R effects and receptor regulation is unclear. CB(1)R signaling and behaviors (antinociception, hypothermia, catalepsy) were assessed in β-arrestin2-knockout (βarr2-KO) and wild-type mice after THC administration. Cannabinoid-stimulated [(35)S]GTPγS and [(3)H]ligand autoradiography were assessed by statistical parametric mapping and region-of-interest analysis. β-arrestin2 deletion increased CB(1)R-mediated G-protein activity in subregions of the cortex but did not affect CB(1)R binding, in vehicle-treated mice. βarr2-KO mice exhibited enhanced acute THC-mediated antinociception and hypothermia, with no difference in catalepsy. After repeated THC administration, βarr2-KO mice showed reduced CB(1)R desensitization and/or downregulation in cerebellum, caudal periaqueductal gray, and spinal cord and attenuated tolerance to THC-mediated antinociception. In contrast, greater desensitization was found in hypothalamus, cortex, globus pallidus, and substantia nigra of βarr2-KO compared with wild-type mice. Enhanced tolerance to THC-induced catalepsy was observed in βarr2-KO mice. β-arrestin2 regulation of CB(1)R signaling following acute and repeated THC administration was region-specific, and results suggest that multiple, overlapping mechanisms regulate CB(1)Rs. The observations that βarr2-KO mice display enhanced antinociceptive responses to acute THC and decreased tolerance to the antinociceptive effects of the drug, yet enhanced tolerance to catalepsy, suggest that development of cannabinoid drugs that minimize CB(1)R interactions with β-arrestin2 might produce improved cannabinoid analgesics with reduced motor suppression.

Regional enhancement of cannabinoid CB1 receptor desensitization in female adolescent rats following repeated Δ9‐tetrahydrocannabinol exposure

Disruption of the substantial re-organization of the brain during adolescence may be induced by persistent abuse of marijuana. The aim of this study was to determine whether adolescent and adult rats exhibit differential adaptation of brain cannabinoid (CB(1)) receptors after repeated exposure to Delta(9)-tetrahydrocannabinol (THC). Rats of both ages and sexes were dosed with 10 mg kg(-1) THC or vehicle twice daily for 9.5 days. Subsequently, CB(1) receptor function and density were assessed. In all brain regions, THC treatment produced desensitization and down-regulation of CB(1) receptors. While the magnitude of down-regulation did not differ across groups, greater desensitization was evident in the brains of THC-treated female adolescent rats for most regions. Adolescent females showed greater desensitization than adult females in the prefrontal cortex, hippocampus, periaqueductal gray (PAG) and ventral midbrain. In contrast, adolescent males exhibited less desensitization in the prefrontal cortex, hippocampus and PAG, an effect opposite to that seen in females. With the exception of the PAG, sex differences were seen only in adolescents, with greater desensitization in the prefrontal cortex, striatum, hippocampus, PAG, and ventral midbrain of females. These results suggest that the brains of adolescent females may be particularly vulnerable to disruption of CB(1) receptor signalling by marijuana abuse. Alternatively, increased desensitization may reflect protective adaptation. Given the extensive re-organization of the brain during adolescence, this disruption has potential long-term consequences for maturation of the endocannabinoid system.

Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment

To investigate effects of altering troponin (Tn)C Ca(2+) binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca(2+) sensitivities. Ca(2+) binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys(99) residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca(2+) sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca(2+) dissociation rate (k(off)), respectively. On the basis of K(d) and k(off), these results suggest that the Ca(2+) association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca(2+) dependence of force development and rate of force redevelopment (k(tr)) at 15°C, resulting in sensitization of both force and k(tr) to Ca(2+) for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca(2+), I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (F(max)) and maximal k(tr). Although V43QTnC and I60QTnC had drastically different effects on Ca(2+) binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated k(tr) at force levels <30%F(max) vs. wild-type TnC. However, at matched force levels >30%F(max) k(tr) was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected k(tr) through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal k(tr) for I60QTnC at low Ca(2+) concentration. Thus TnC mutants with altered Ca(2+) binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.

Phosphorylation promotes the desensitization of the opioid-induced Ca 2+ increase in NG108-15 cells

Using the fluorescent Ca 2+ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca 2+ concentration ([Ca 2+] i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca 2+] i increase, while exposure of desensitized cell to 50 mM K + restores the response EK-induced, suggesting a role of intracellular Ca 2+ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[ N, O-bis-(5-isoquinolinesulfonyl)- N-methyl- l-tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4,5-trisphosphate (IP 3) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP 3 generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca 2+ influx by either verapamil or removal of extracellular Ca 2+ or the depletion of Ca 2+ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca 2+ transporters involved in Ca 2+ influx and Ca 2+ release. Our results imply that emptying of intracellular Ca 2+ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.

Desensitization in normal and neoplastic human thyroid cell lines.

Because some papillary thyroid cancers continue to grow when thyroid-stimulating hormone (TSH) levels are suppressed, we questioned whether desensitization (i.e., a decreased cAMP response to repeat stimulation with TSH) occurs in normal and neoplastic thyroid tissue. If desensitization does occur, is it similar or different in these human thyroid cells? Normal and papillary thyroid cancer cells from the same patient were cultured as we have previously described. Normal and neoplastic thyroid tissues responded to TSH (0.01-10.0 mU/ml) by increasing cAMP production and growth in a dose-dependent manner. In normal cells there was an 11-fold mean increase in cAMP production at 4 hours, and all thyroid cultures responded. In neoplastic cells cAMP production increased from 1.5-fold to 3.0-fold with a mean 2.0-fold increase at 4 hours. In normal thyroid cells the cAMP response to a second TSH stimulus (desensitization) decreased up to 75% (range 25-75%), and desensitization occurred in all normal thyroid cell cultures. In neoplastic thyroid cells, however, the cAMP response to a second TSH stimulus decreased up to 17% (range 0-17%); and desensitization occurred in only two of the five neoplastic thyroid cell cultures. Thus when normal thyroid and neoplastic cells from the same patients were studied, greater desensitization occurred in the normal cells (75% vs. 17%). These studies document that there is greater desensitization in normal tissue than in neoplastic thyroid tissue, which may account for the increased growth of thyroid neoplasms in the presence of ever-changing low levels of TSH.

On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea‐pig atrial cells.

1. The ACh-activated K+ current (IK,ACh) has been investigated in guinea-pig atrial cells at 36 degrees C using the whole-cell patch-clamp technique. 2. During an exposure to ACh, IK,ACh faded as a result of desensitization. Throughout the fade of the current, the current reversed at EK and showed inward-going rectification. The fade was, therefore, the result of a genuine decrease in IK,ACh. 3. The onset of desensitization (as judged by the fade of IK,ACh) was biphasic and the time constants of the fast and slow phases of desensitization were 1.58 +/- 0.14 (n = 16) and 148.2 +/- 12.8 s (n = 18) respectively. Recovery from the fast and slow phases of desensitization (after 30 s and 5 min exposures to ACh respectively) occurred with time constants of 52 and 222 s respectively. This suggests that two processes are involved in desensitization. 4. The Q10 of the rate constant of the fast phase of desensitization was 2.2 +/- 0.3 (n = 6). 5. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or extracellular perfusion with AlF4- were used to bypass the muscarinic receptor and trigger IK,ACh by directly activating the G protein, GK, that links the muscarinic receptor to the K+ channel. Both GTP gamma S and AlF4- activated a current with the same reversal potential and the same degree of inward-going rectification as the ACh-activated current. 6. Desensitization still occurred when the muscarinic receptor was bypassed and IK,ACh was triggered by direct activation of GK with either GTP gamma S or AlF4-. This suggests that desensitization is, in part, the result of a modification of either GK or the K+ channel. 7. Activation of the muscarinic receptor by ACh resulted in greater desensitization than direct activation of GK; at the end of a 5 min exposure to ACh, current was only 22 +/- 1% (n = 19) of its peak value, whereas, after direct activation of GK by GTP gamma S for 5 min, current was 42 +/- 6% (n = 5) of its peak value. This suggests that desensitization also involves the muscarinic receptor. 8. When cells were perfused with GTP gamma S, the fast phase of desensitization could still occur, but the slow phase was reduced. This suggests that the fast phase involves GK or the K+ channel, whereas the slow phase involves the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization of β-Adrenoceptor-Mediated Functional Responses of Guinea Pig Atria by In Vitro Incubation with Isoprenaline

Positive inotropic and chronotropic responses to (-)-isoprenaline were recorded in guinea pig isolated paced left and spontaneously beating right atria. Concentration-response curves were determined before and after incubation with 10(-6) M (-)-isoprenaline for various times from 0.5 to 8 h. Before construction of the postincubation curve, isoprenaline was washed from the tissue for either 0.5 or 1 h. Preincubation curves were corrected from controls for time-dependent changes in sensitivity. After a 2-h incubation with 10(-6) M isoprenaline and a 0.5-h washout, there was a significant rightward shift of the right atrial rate curve [concentration ratio (CR) 10.6 +/- 2.1]. This was reversed by extending the washout to 1 h. After a 4-h incubation, however, a poorly reversible desensitization occurred, the curve still being significantly shifted after a 1-h washout (CR, 17.4 +/- 6.2). A higher incubation concentration, 10(-5) M, appeared to produce greater desensitization as a depression of the maximum response, however, this was an artifact caused by the failure to wash the isoprenaline from the tissue. When left atria were paced throughout the incubation period, a substantial nonspecific depression of the maximum response to 73.7 +/- 7.6% occurred. When pacing was stopped during incubation, only a rightward shift of the curve was produced by incubation with isoprenaline (10(-6) M), with no change of maximum (96.5 +/- 6.3%). There was no cross-desensitization of histamine in either the left (unpaced while incubating) or right atria after a 4-h incubation period with isoprenaline (10(-6) M). The desensitization was therefore beta-adrenoceptor specific and of the homologous type. After an 8-h incubation, there was an additional depression of the postincubation maximum which was greater in the left (68.9 +/- 2.3%) than right atria (85.2 +/- 5.4%). This true reduction of the maxima indicates that desensitization had effectively removed a proportion of the beta adrenoceptors.

Cholecystokinin-induced desensitization of pepsinogen secretion from chief cells

When dispersed chief cells from guinea pig stomach were first incubated with cholecystokinin (CCK), washed, and then reincubated with CCK in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. CCK did not cause residual enzyme secretion but caused desensitization that was rapid, temperature dependent, dependent on extracellular calcium, reversible with time, and prevented but not reversed by CCK receptor antagonists. Cholecystokinin octapeptide (CCK-8) caused desensitization over the same range of concentrations that stimulate pepsinogen secretion, whereas the concentration of gastrin required to cause maximal desensitization was greater than that required to cause maximal stimulation of enzyme secretion. CCK-8 caused heterologous desensitization of pepsinogen secretion stimulated by agonists that interact with receptors to cause mobilization of cellular calcium and activation of protein kinase C or by agonists that bypass receptors to activate these mediators directly; however, CCK-8 did not induce desensitization of the stimulation caused by any secretagogue whose actions are mediated by adenosine 3',5'-cyclic monophosphate. Because CCK-8 caused greater desensitization of secretion stimulated by agonists that interact with receptors than by agonists that bypass receptors, it is likely that receptor modulation as well as a postreceptor action contribute to the ability of CCK to cause desensitization of pepsinogen secretion from chief cells.

Desensitization to acetylcholine at motor end plates in normal humans, patients with myasthenia gravis, and experimental models of myasthenia gravis.

Desensitization to ionophoretically applied ACh at the motor end plates of normal humans and patients with MG was studied. The pattern of desensitization at the normal human motor end plate was completely consistent with that observed in muscles of various animals. At motor end plates of MG patients there was greater desensitization and slower recovery from desensitization than in normals. A similar pattern of desensitization was observed at normal end plates when they were exposed to serum globulins of MG patients, and at end plates of muscles from mice than were repeatedly injected with serum globulins of MG patients. Despite the reported reduction in the number of ACh receptors, our calculations show that there is a large pool of spare receptors forming a sizeable margin of safety at the end plates of MG patients. The number of receptors may not show additional progressive reduction in number during repetitive motor activity. Our results indicate that desensitization could play a significant role in the development of neuromuscular block following repetitive motor activity in MG patients. The mechanism by which desensitization is augmented in MG patients in not yet clear.