Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.     

Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.     

Effects of selective α1A-adrenoceptor antagonists on reperfusion arrhythmias in isolated rat hearts

Stimulation of ₁-adrenoceptors (AR) during ischaemia in the rat heart by exogenous phenylephrine exacerbates reperfusion arrhythmias, an effect apparently mediated by the _1A-AR subtype. We tested whether _1A-AR stimulation byendogenous catecholamines, released during ischaemia, could modulate reperfusion arrhythmias, using as pharmacological tools the selective _1A-AR antagonists abanoquil (UK52046) and WB4101. Isolated rat hearts (n=12/group) were subjected to dual coronary perfusion. After 15 min of aerobic perfusion of both coronary beds, abanoquil or WB4101 was infused selectively into the left coronary bed (LCB) for 5 min. The LCB was then subjected to 10 min of zero-flow ischaemia and 5 min of reperfusion. Effects on PR interval, width of the ventricular complex (QRST₉₀) and reperfusion arrhythmias were assessed. Abanoquil at concentrations of 0.03, 0.1 and 0.3 M tended to reduce the incidence of reperfusion-induced ventricular fibrillation (VF) in a dose-dependent manner from 75% in controls to 58, 33 and 25%, but this effects did not achieve statistical significance. Similarly, WB4101 at 0.1, 0.3 and 1 M also tended to reduce VF incidence from 67% in controls to 67, 42% and 33% (NS). The incidence of ventricular tachycardia (VT) was 100% in all groups and ECG parameters were not altered significantly by either drug. These results suggest that, in this denervated isolated heart preparation, _1A-AR stimulation during ischaemia by endogenous catecholamines does not significantly modulate reperfusion arrhythmias.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat

Adriamycin is one of the most effective and useful antineoplastic agents. Acute doxorubicin cardiotoxicity involved cardiomyocyte apoptosis. In this study, we investigated whether adriamycin induced myocardium apoptosis through activation of nuclear factor κB in rat. Forty male Wistar rats were randomly divided into five groups: control, ADR 5 mg/kg, ADR 10 mg/kg, ADR 15 mg/kg group and ADR + PDTC 200 mg/ml group. Myocardial apoptosis was detected by DNA fragmentation assay and TUNEL assay; Location and distribution of p-IκBα was observed by immunohistochemical assay; Myocardial expression of p-IκBα protein was assessed by Western blot analysis; Activity of NF-κB was evaluated by Electrophoretic Mobility Shift Assay. The myocardial apoptotic index, expression of p-IκBα, and binding activity of NF-κB increased significantly in ADR groups in dose-dependent manner. PDTC as a nonspecific inhibitor of NF-κB protected myocardium from apoptosis by inhibiting NF-κB activation. Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat and NF-κB activation requires IκBα degradation.     

Shaping immune responses through the activation of dendritic cells–P2 receptors

Dendritic cells (DCs) activate and shape the adaptive immune response by capturing antigens, migrating to peripheral lymphoid organs where naïve T cells reside, expressing high levels of MHC and costimulatory molecules and secreting cytokines and chemokines. DCs are endowed with a high degree of functional plasticity and their functions are tightly regulated. Besides initiating adaptive immune responses, DCs play a key role in maintaining peripheral tolerance toward self-antigens. On the basis of the information gathered from the tissue where they reside, DCs adjust their functional activity to ensure that protective immunity is favoured while unwanted or exaggerated immune responses are prevented. A wide variety of signals from neighbouring cells affecting DC functional activity have been described. Here we will discuss the complex role of extracellular nucleotides in the regulation of DC function and the role of P2 receptors as possible tools to manipulate immune responses.     

Preparation of 123I-labeled 2′-iodospiperone and imaging of D2 dopamine receptors in the human brain using SPECT

[¹²³I]2′-ISP was readily prepared using a radioiodine exchange reaction with a radiochemical yield of approx. 50% after HPLC purification. The radiochemical purity of the product was more than 98% and the specific activity was 5.55–11.1 GBq/μmol. Biodistribution studies performed in mice indicated that injection of [¹²³I]2′-ISP with albumin produced a higher gastric uptake and a lower brain uptake than injection of the radioligand in a weakly acidic solution. In addition, toxicity tests performed in mice demonstrated that acute toxic effects would be very unlikely to be encountered if 2′-ISP was used for diagnostic purposes. A preliminary imaging study with [¹²³I]2′-ISP in a healthy human volunteer showed its specific uptake by the basal ganglia, a region of the brain known to have a high density of D₂ dopamine receptors.     

Involvement of Arabidopsis thaliana phospholipase Dζ2 in root hydrotropism through the suppression of root gravitropism

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dζ2 gene (PLDζ2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldζ2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldζ2 mutant roots. In root hydrotropism experiments, pldζ2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDζ2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDζ2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.     

Antinociceptive effects of novel epibatidine analogs through activation of α4β2 nicotinic receptors

The study of α4β2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4β2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4β2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury(CCI)-induced neuropathic pain in rats, which was sensitive to DHβE, a selective α4β2 subtype antagonist,indicating that its analgesic effect was mediated by the activation of the α4β2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4β2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

Attenuation of ischemia–reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts

Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.     

Inhibition of associative long-term depression by activation of β-adrenergic receptors in rat hippocampal CA1 synapses

The aim of this study was to investigate the role of β-adrenergic receptors in modulating associative long-term depression (LTD) at CA1 synapses in rat hippocampal slices. Standard extracellular electrophysiological techniques were employed to record field excitatory post-synaptic potential (fEPSP) activity and to induce associative LTD. Two independent Schaffer collateral pathways were elicited in hippocampal CA1 areas. In one (weak) pathway, the stimulating intensity was adjusted to elicit small fEPSP activity (20–30% of the maximum response). In contrast, 80–90% of the maximum response was evoked in the other (strong) pathway. Associative LTD of weak pathway could be induced by paired stimulation of weak and the strong pathways, repeated 100 times at 0.167 Hz. The associative LTD of weak pathway was NMDA receptor- and phophatase 2B dependent, because bath application of 50 μM D, L-AP5 or 10 μM cypermethrin blocked its induction. Bath application of 1 μM isoproterenol inhibited associative LTD, and this effect was blocked by timolol, suggesting the involvement of β-adrenergic receptors. The inhibitory effect of β-adrenergic receptors on LTD induction was blocked in slices pretreated with inhibitors of protein kinase A and mitogen-activated protein kinase, suggesting that these signal cascades are downstream effectors following activation of β-adrenergic receptors. Nevertheless, bath application of timolol or cypermethrin alone did not have significant effect on associative LTD induction, suggesting neither endogenous function of β-adrenergic receptor nor endogenous PKA activity does have a role in associative LTD induction.     

Acylated and unacylated ghrelin attenuate isoproterenol-induced lipolysis in isolated rat visceral adipocytes through activation of phosphoinositide 3-kinase γ and phosphodiesterase 3B

The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic β-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3Kγ) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser(473). Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3Kγ/AKT and PDE3B.     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9α, 11β-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D₂ was investigated in isolated perfused rat lung. Dose-dependent formation (2–150 ng) of 9α,11β-prostaglandin F₂, corresponding to about 0.1% of the perfused dose of prostaglandinD₂, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9α,11β-prostaglandin F₂ and prostaglandin D₂ by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9α,11β-prostaglandin F₂ disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ by GC/MS. In contrast, pulmonary breakdown of prostaglandin D₂ was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D₂ and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D₂ in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9α,11β-prostaglandin F₂ formed is rapidly metabolized to 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂.     

Isomers of conjugated linoleic acid induce insulin resistance through a mechanism involving activation of protein kinase Cε in liver cells

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser⁴⁷³ and Thr³⁰⁸, the insulin receptor at Tyr¹¹⁵⁸, IRS-1 at Tyr⁶³², and GSK-3 at Ser^9/21. Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser⁶¹², a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation.     

Lack of interaction between α2 and dopamine D2-receptors in mediating their inhibitory effects on [3H]dopamine release from rat nucleus accumbens slices

The ₂-adrenoceptor agonist, UK14304, dose-dependently inhibited the electrically stimulated release of dopamine (DA) from rat nucleus accumbens slices. This effect was antagonized by idazoxan, confirming that it was an ₂-adrenoceptor mediated effect. There was no evidence of endogenous activation of noradrenergic receptors suggesting that the ₂-adrenoceptor agonist was not acting presynaptically to inhibit noradrenaline release. An in vitro superfusion technique was used to investigate wheher there was any interaction between ₂-adrenoceptors and DA D₂-receptors in mediating their inhibitory effects on [³H]DA release from rat nucleus accumbens slices. ₂-Adrenoceptor and DA D₂-receptors interact with similar second messenger systems and it was considered that they may compete for a common pool of G-proteins. The inhibitory effects of the ₂-adrenoceptor agonist, UK14304, and the DA receptor agonists, quinpirole, apomorphine and pergolide were not independent. However, there was no evidence of any interaction between UK14304 and the DA D₂-receptor antagonists, sulpiride or haloperidol, suggesting that the two receptors do not compete for a common pool of G-proteins in mediating their inhibitory effects on DA release.     

Role of negatively charged amino acids in β4 F-loop in activation and desensitization of α3β4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the β4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the α3β4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at − 40 mV produced inward currents which displayed biphasic desensitization. The EC₅₀ of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC₅₀ increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.