Wheat germ agglutinin behaves as a GnRH antagonist but induces gonadotrope desensitization

Preincubation of cultured rat pituitary cells with 10 micrograms/ml of either wheat germ agglutinin (WGA) or concanavalin A inhibited LH release stimulated with GnRH (0.5 nM) by 55% and 40%, respectively. WGA-inhibition of LH release stimulated by GnRH was dose-dependent, reaching a plateau of 75% inhibition at 50 micrograms/ml. Concomitantly, WGA induced a dose-dependent inhibition of 125I-Buserelin specific binding to pituitary cells, with a maximal inhibition of 45%. The inhibition of 125I-Buserelin binding by WGA is due to GnRH receptor internalization and not to persistent occupancy of the receptors. In addition to the effect of WGA on receptor internalization, WGA also induced partial desensitization of pituitary cells but was ineffective in modulating GnRH-induced desensitization. These findings indicate that WGA has all the characteristics of a GnRH antagonist, nevertheless, it does induce desensitization of cultured rat pituitary cells to further stimulation with GnRH.     

Gonadotropin-releasing hormone analogs inhibit primate granulosa cell steroidogenesis via a mechanism distinct from that in the rat

Gonadotropin-releasing hormone (GnRH) and related peptides are implicated in the local control of rat ovarian function, but evidence to date for direct effects of such peptides on primate ovarian cells is equivocal. In contrast to rat ovaries, where GnRH action is mediated through specific, high-affinity GnRH receptors, no such binding sites have been identified in primate tissue. Using undifferentiated granulosa cells from immature follicles in cyclic (luteal phase) marmoset ovaries, we have observed direct suppression of human (h) FSH-induced steroidogenesis by GnRH analogs in vitro. Granulosa cells from immature (less than 1 mm diameter) follicles were incubated for 4 days in the presence of hFSH and testosterone (aromatase substrate) to stimulate cyclic AMP (cAMP) production and steroidogenesis. The additional presence of GnRH alone (up to 10 microM) had no effect on FSH action. However, the GnRH agonist, [D-Ser(But)6]GnRH 1-9)-ethylamide (Buserelin, 0.1 microM-10 microM), caused time- and dose-dependent inhibition of estradiol (maximum inhibition = 79%; ED50 = 0.55 microM) and progesterone production (maximum inhibition = 93%; ED50 = 0.1 microM). Accumulation of cAMP was also inhibited by up to 54%. Paradoxically, a GnRH antagonist [( N-Ac-D-Nal(2)1,D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10]-GnRH; 10 microM) alone also inhibited hFSH-stimulated cAMP and steroid production by 40% and 70%, respectively. Moreover, the suppressive effects of the GnRH agonist on granulosa cell functions were augmented by the presence of the GnRH antagonist (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of myometrial desensitization to beta-adrenergic agonists

Continuous exposure of ovine myometrial strips exposed to isoproterenol (10 microM) resulted in only transient inhibition with contractions returning within 60 min. Rechallenging these strips with isoproterenol failed to induce inhibition, confirming the occurrence of desensitization. In contrast, exposure of myometrial tissue to isoproterenol for only 5 min did not result in desensitization. Myometrial strips exposed to isoproterenol demonstrated a significant increase in cAMP content associated with inhibition of contractile activity and a subsequent fall in cAMP content upon desensitization. Elevation of endogenous cAMP levels by either inhibition of cAMP-dependent phosphodiesterase activity (0.5 mM isobutylmethylxanthine, in ovine strips) or direct activation of adenylyl cyclase (10 microM forskolin, in rat strips) induced a rapid and significant inhibition of myometrial contractile activity in desensitized tissue. Scatchard analysis of the binding of the beta-adrenoceptor antagonist, [125I]iodocyanopindolol, revealed a significant reduction in the concentration of beta-adrenergic receptors (but no change in binding affinity) in desensitized myometrial tissue. Incubation of desensitized tissue with fresh buffer for 3 h induced only a partial recovery in responsiveness to isoproterenol. These data suggest that prolonged, but not acute, exposure of the myometrium to beta-adrenergic agonists induces a state of desensitization that is associated with a down-regulation of beta-adrenoceptors but maintenance of postreceptor function.     

Copper induces luteinizing hormone release and desensitization of pituitary gonadotropes

Copper stimulated LH release from cultured rat pituitary cells in a dose-and time-dependent manner. After 4 h of incubation with 10 mu M Cu2+, LH release was stimulated by 3-fold. The release of LH stimulated by Cu2+ was Ca2+ dependent, thus excluding the possibility that the releasing activity of this divalent cation was due to a toxic effect on pituitary cells. The stimulatory action of Cu2+ is substantially mediated via the GnRH-receptors since Cu2+ inhibited 125I-Buserelin binding and since GnRH-antagonist blocked most of the Cu2+-stimulated LH release (80%). Both GnRH (1 microM) and Cu2+ (10 microM) induced desensitization of pituitary cells to a subsequent stimulation of either GnRH (0.5 nM) or Cu2+ (10 microM). However, in contrast to GnRH, Cu2+ did not induce down regulation of GnRH receptors. These findings suggest that the Cu2+ effects are mainly mediated through the GnRH receptors.     

Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values.

Treatment of intact hepatocytes with glucagon led to the rapid desensitization of adenylate cyclase, which reached a maximum around 5 min after application of glucagon, after which resensitization ensued. Complete resensitization occurred some 20 min after the addition of glucagon. In hepatocytes which had been preincubated with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), glucagon elicited a stable desensitized state where resensitization failed to occur even 20 min after exposure of hepatocytes to glucagon. Treatment with IBMX alone did not elicit desensitization. The action of IBMX in stabilizing the glucagon-mediated desensitized state was mimicked by the non-methylxanthine cyclic AMP phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxylbenzyl)-2-imidazolidinone]. IBMX inhibited the resensitization process in a dose-dependent fashion with an EC50 (concn. giving 50% of maximal effect) of 26 +/- 5 microM, which was similar to the EC50 value of 22 +/- 6 microM observed for the ability of IBMX to augment the glucagon-stimulated rise in intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with IBMX did not alter the ability of either angiotensin or the glucagon analogue TH-glucagon, ligands which did not increase intracellular cyclic AMP concentrations, to cause the rapid desensitization and subsequent resensitization of adenylate cyclase. It is suggested that, although desensitization of glucagon-stimulated adenylate cyclase is elicited by a cyclic AMP-independent process, the resensitization of adenylate cyclase can be inhibited by a process which is dependent on elevated cyclic AMP concentrations. This action can be detected by attenuating the degradation of cyclic AMP by using inhibitors of cyclic AMP phosphodiesterase.     

Mechanisms for the antigonadotropic action of the ovarian gonadotropin-releasing hormone-binding inhibitor protein/histone H2A on ovarian cells.

A GnRH-binding inhibitor (GnRH-BI) was recently purified from bovine ovaries. On the basis of amino acid composition and partial sequence analysis this antigonadotropic GnRH-BI was identified as histone H2A. In the present study the mechanism for the antigonadotropic action of histone H2A was examined and compared to that of GnRH and poly-L-lysine. The potential sites examined were the receptor-coupled pathway of second message synthesis including receptor binding of hormone, G protein activation, and adenylyl cyclase activation. Histone H2A inhibited (ID50 = 2 microM) the binding of hCG by membrane receptors from luteinized rat ovaries in a noncompetitive and dose-dependent manner. The binding of FSH by membrane receptors from immature rat ovaries was not inhibited by histone H2A. Binding of GnRH by pituitary membrane receptors was inhibited by histone H2A, and the ID50 of 8 microM was similar to that previously observed for GnRH binding sites in rat ovarian membranes. No high-affinity binding of histone H2A by rat ovarian membranes was detected. Near-maximal doses of histone H2A (7 microM), poly-L-lysine (10 microM), and GnRH (1 microM) inhibited LH-stimulated cAMP production in isolated rat luteal cells. Inhibition by H2A and poly-L-lysine was larger than by GnRH. Furthermore, histone H2A and poly-L-lysine inhibited cholera toxin (CT)-stimulated cAMP production, but GnRH did not. Like GnRH, neither histone H2A nor poly-L-lysine inhibited forskolin (FK)-stimulated cAMP production. In isolated rat granulosa cells, histone H2A and poly-L-lysine inhibited FSH-, CT-, and FK-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons

Two alpha-bungarotoxin-sensitive nicotinic receptor subtypes in cockroach neurons are identified as desensitizing (nAChD), selectively inhibitable with 100 nM imidacloprid, and non-desensitizing (nAChN), selectively inhibitable with 100 pM methyllycaconitine. Although the desensitization rate of nAChD receptors is highly variable, pharmacology is largely independent of desensitization rate. Because desensitized states tightly bind agonists, nAChD receptors are potently inhibited by neonicotinoids and specifically measured in radiolabeled imidacloprid binding assays. However, they are not usually detected in binding assays with radiolabeled alpha-bungarotoxin, which has a Kd for the resting state of 21 nM, but binds poorly to desensitized states often present in binding assays. In contrast, nAChN receptors are specifically measured in binding assays with radiolabeled alpha-bungarotoxin, which binds them with a Kd of 1.3 nM. nAChN receptors are activated by neonicotinoids at micromolar concentrations, and allosterically by spinosyn A, with an EC50 of 27 nM. Spinosyn A weakly antagonizes nAChD receptors -23% at 10 microM. The roles of the two nAChR subtypes in insecticide poisoning are discussed.     

Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.     

Desensitization of contact allergy to DNFB in mice. I. Description of a model system

We investigated the down-regulation of contact sensitivity (desensitization) in mice sensitized to DNFB. Mice were sensitized with DNFB, desensitized with antigen 2 wk later, and resensitized 2 wk after desensitization. Large doses of antigen (DNFB or DNBSO3) produced about 50% inhibition of the anamnestic response as measured by ear swelling after challenge with DNFB. Desensitization was antigen specific and long lasting. Lymph node cells from desensitized mice showed diminished antigen-induced proliferation in vitro. Although the anamnestic response can be inhibited by afferent- or efferent-acting suppressor cells, such suppressor cells were not demonstrated in desensitized animals. The most likely explanation is that antigen desensitizes by inactivating effector cells for contact sensitivity, although suppressor mechanisms have not been completely excluded.     

Homologous desensitization with gonadotropin-releasing hormone (GnRH) also diminishes gonadotrope responsiveness to maitotoxin: a role for the GnRH receptor-regulated calcium ion channel in mediation of cellular desensitization

Maitotoxin (MTX) stimulates gonadotropin release from pituitary cell cultures. The time course and efficacy of LH release in response to GnRH and to MTX are similar; both secretagogues require extracellular Ca2+ and are inhibited by the selective Ca2+ ion channel antagonist methoxyverapamil (D600). LH release in response to either GnRH or MTX is not measurably inhibited by two other chemical classes of Ca2+ ion channel inhibitors represented by nifedipine and by diltiazem. The two secretagogues are nonadditive in their action on LH release when presented at high doses and prior studies indicate that MTX has no endogenous ionophoretic activity. These observations indicate that MTX likely stimulates LH release due to activation of the GnRH receptor associated Ca2+-ion channel in the gonadotrope. We have therefore assessed the functional state of this channel during the development of homologous desensitization of the gonadotrope to GnRH by measuring the ability of MTX to stimulate LH release. Cells were desensitized with GnRH in the presence of 3 mM EGTA. Under these conditions, the cells become refractory to GnRH in the absence of gonadotropin release since the latter process, but not the former, requires extracellular Ca2+. Accordingly, this approach allows assessment of the degree of desensitization in the absence of the influence of gonadotropin depletion. Such desensitized cells are less responsive to GnRH. Desensitized pituitary cells also respond with diminished efficacy and potency to MTX three or more hours after GnRH treatment but not at an earlier time (1 h) when GnRH receptors are diminished. These data are consistent with a model in which homologous desensitization is viewed as developing in two phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.     

Cycloheximide induces accumulation of vasoactive intestinal peptide (VIP) binding sites at the cell surface of a human colonic adenocarcinoma cell line (HT29-D4). Evidence for the presence of an intracellular pool of VIP receptors

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

On the mechanism of isoproterenol-induced desensitization of adenylate cyclase in cultured differentiated hepatocytes

The adenylate cyclase of cultured differentiated RL-PR-C hepatocytes is desensitized to 1-isoproterenol by exposure to this beta-agonist. Virtually complete desensitization occurred by 60 min (intact cells) or 30 min (isolated plasma membranes). Isoproterenol was maximally effective at 10 micrometers, although substantial desensitization occurred at isoproterenol concentrations as low as 10 nM. Protein synthesis was not required for desensitization. Recovery from desensitization under tissue culture conditions was only 25% complete by 24 h. Maximum desensitization was accompanied by only a modest 35% decrease in binding sites (as determined by binding assays with [3H]dihydroalprenolol), with no change in binding affinity. Adenylate cyclase desensitized to 1-isoproterenol responded normally to guanine nucleotides and to fluoride, suggesting that the regulatory and catalytic proteins were not the sites of the desensitization "defect'. Using N-ethylmaleiimide to inactive the regulatory and catalytic proteins, and dicyclohexylcarbodiimide to inactivate the beta-adrenergic receptor, of intact hepatocytes, various heterologous cell fusion hybrids were produced, and their adenylate cyclases tested for responsiveness to 1-isoproterenol; only hybrids containing "desensitized' receptor failed to respond to isoproterenol. These results suggest that the mechanism of desensitization to isoproterenol involves only the receptor component of the receptor-regulatory protein(s)-adenylate cyclase complex, and that the receptors are reduced in number and/or ability to interact with the regulatory protein as a result of the desensitization process.     

Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.     

Desensitization of contract allergy to 2,4-dinitro-1-fluorobenzene in mice. II. Characteristics of "immediate" desensitization

The immediate effects and mechanisms of desensitization of contact sensitivity to 2,4-dinitro-1-fluorobenzene (DNFB) were investigated. Mice were sensitized with DNFB, desensitized with antigen 2 weeks later, and challenged 1 day after desensitization. Significant inhibition (approximately 50%) of contact sensitivity was observed after iv injections of large doses of dinitrobenzene sulfonic acid (DNBS) or dinitropenol (DNP)-labeled spleen cells. Haptenated red blood cells (RBC) did not induce any significant immediate desensitization but produced significant inhibition of an anamnestic response 2 weeks later. The immediate desensitization induced by DNBS was antigen nonspecific. Although the contact sensitivity response itself could be inhibited by afferent- or efferent-acting suppressor cells, such cells were not demonstrated in desensitized animals. DNBS appears to desensitize by inactivating effector cells for contact sensitivity, although it appears that suppressor mechanisms could be activated by other physiochemical forms of the desensitizing antigen.     

Rapid desensitization and resensitization of 5-HT2 receptor mediated phosphatidyl inositol hydrolysis by serotonin agonists in quiescent calf aortic smooth muscle cells

Agonist regulation of 5-hydroxytryptamine2 (5-HT2) receptors was studied in calf aortic smooth muscle cultures incubated in a quiescent, defined synthetic medium that does not stimulate cell proliferation, but that provides cells with supplements that maintain cell viability. In these cells, 5-hydroxytryptamine (5-HT)-induced [3H]inositol phosphates accumulation showed the characteristics of a 5-HT2 receptor coupled transducing system according to the inhibition of the response by 5-HT2 antagonists at nanomolar concentrations. The 5-HT2 receptor coupled response became rapidly desensitized during continued incubation with 5-HT and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); nearly full desensitization was obtained in two hours with 10 microM 5-HT and DOM pretreatment. The recovery of the response had a half-live of 5 hours after 2 hours pretreatment and of 9.5 to 12.5 hours after 24 to 96 hours agonist pretreatment. The DOM-induced desensitization of the 5-HT2 receptor coupled response was fully blocked by 0.1 microM cinanserin. Cinanserin alone did not induce desensitization or up-regulation of the 5-HT2 receptor coupled response at 0.1 microM. It may be that the down-regulation of central 5-HT2 receptors by antagonists in vivo is a heterologous process due to mediators which are triggered by 5-HT2 antagonistic action.     

Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPR_des) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPR_des back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPR_des as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPR_des also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPR_des initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.