Use of bioluminescent aequorin for the pharmacological characterization of 5HT receptors.

A convenient functional assay for 5HT2a and 5HT2c receptors is reported utilizing the bioluminescent aequorin to detect intracellular calcium changes. Using this assay, the pharmacological properties of many 5HT ligands can be determined in a 96-well format. The data indicate that the aequorin detection method is superior to the inositol phosphate assay with regard to speed and scope. This system is also appropriate for kinetic studies of receptor desensitization. We showed that the human 5HT2c receptor desensitizes in a biphasic manner, with a fast desensitization of approximately 90% of the total response occurring within 15 minutes while the remaining 10% response remains for at least 3 hours.     

Thyrotrophin Releasing Hormone - 5-Hydroxytryptamine Interactions in the Brain Studied Using Chronic Immunization and Chemical Lesioning Techniques

Abstract

This study investigates the effects of chemically lesioning 5-hydroxytryptamine (5HT) neurones and chronic passive immunization of central thyrotrophin releasing hormone (TRH) on 5HT and TRH mediated behavioural responses. 5HT lesions produced by 5,7-dihydroxytryptamine (5,7-DHT) enhanced the behavioural response produced by the 5HT receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MEODMT) while decreasing the locomotor hyperactivity observed following administration of the TRH analogue CG 3509 but having no effect on the reversal of pentobarbitone sleep-time produced by CG 3509. Chronic intracerebroventricular infusion of the purified TRH antibody markedly increased the length of pentobarbitone-induced sleep-time while enhancing the effects of CG 3509 both on locomotor activity and pentobarbitone-induced sleep. TRH antibody infusion also increased the response produced by 5-MEODMT. The results indicate that chronic passive immunization of central TRH induces changes in TRH receptor responsiveness and that there is a functional interaction between TRH and 5HT neuronal systems.     

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y₂ receptors in both 1321N1 cell transfectants expressing the P2Y₂ receptor and human HT-29 epithelial cells expressing endogenous P2Y₂ receptors with an EC₅₀ of 0.2- 1.0 M. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC₅₀ for UTP-induced receptor desensitization was 0.3 - 1.0 M for both systems). Desensitization and down-regulation of the P2Y₂ nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y₂ receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,₂ receptor. In these cells, potent P2Y₂ receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca²⁺]_i, after addition of desensitizing concentrations of UTP, indicating that P2Y₂ receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca²⁺]_i induced by UTP in 1321N1 cell transfectants expressing the P2Y₂ receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y₂ receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y₂ receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y₂ receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y₂ nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y₂ receptor agonists.     

G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.     

Involvement of calmodulin in realization of vasoconstrictive effects of serotonin and norepinephrine

The involvement of calmodulin in adrenergic and serotoninergic regulation of vascular contractility has been studied. Calmodulin inhibitors trifluoperazine and W-13 suppress vasoconstriction of the rat aorta in response to norepinephrine, serotonin, and serotonin 5HT1A and 5HT2A receptor agonists (8-OH-DPAT and DOI, respectively) and do not affect the vasodilatory effect of 5HT1B, 5HT2B, and 5HT4 receptors. The force of aorta contraction in response to 8-OH-DPAT increases after the activation of calcium entry through voltage-gated Ca²⁺ channels. This effect is not related to nonspecific activation of ??1-adrenoceptors, since it is realized in the presence of prazosin. The inhibitor of calmodulin-dependent myosin light chain kinase KN93 decreases the vasoconstrictive response to norepinephrine and serotonin by only 20%. Calmodulin inhibitors slightly decrease aortic constriction in response to endothelin-1, vasopressin, angiotensin II, and KCl. Trifluoperazine does not suppress vasoconstriction induced by the G protein activator AlF ₄ ^? . It is assumed that the target of trifluoperazine and W-13 is calmodulin interacting directly with ??₁-adrenoceptors and serotonin (5HT1A and 5HT2A) receptors.     

Larvae of the small white butterfly,Pieris rapae,express a novel serotonin receptor

The biogenic amine serotonin ( 5‐hydroxytryptamine, 5‐HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G‐protein‐coupled receptors. Five 5‐HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5‐HT_1A, 5‐HT_1B, 5‐HT_2A, 5‐HT_2B, and 5‐HT₇ receptors. We isolated a cDNA (Pr5‐HT₈) from larval Pieris rapae, which shares relatively low similarity to the known 5‐HT receptor classes. After heterologous expression in HEK293 cells, Pr5‐HT₈ mediated increased [Ca²⁺]_i in response to low concentrations (< 10 nM) of 5‐HT. The receptor did not affect [cAMP]_i even at high concentrations (> 10 μM) of 5‐HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5‐HT₈ was also activated by various 5‐HT receptor agonists including 5‐methoxytryptamine, (±)‐8‐Hydroxy‐2‐(dipropylamino) tetralin, and 5‐carboxamidotryptamine. Methiothepin, a non‐selective 5‐HT receptor antagonist, activated Pr5‐HT₈. WAY 10635, a 5‐HT_1A antagonist, but not SB‐269970, SB‐216641, or RS‐127445, inhibited 5‐HT‐induced [Ca²⁺]_i increases. We infer that Pr5‐HT₈ represents the first recognized member of a novel 5‐HT receptor class with a unique pharmacological profile. We found orthologs of Pr5‐HT₈ in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate‐specific receptor because there were no similar receptors in mammals.

     

Functional Receptor Coupling to Gi Is A Mechanism of Agonist-Promoted Desensitization of the β2-Adrenergic Receptor

Abstract

The β₂-adrenergic receptor (β₂AR) couples to G_s, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β₂AR also couple to G_i after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β₂AR-G_i coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β₂AR or β₂AR and G_iα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β₂AR desensitization. Membrane AC activities showed that G_iα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing G_iα2. in the absence of such overexpression, β₂AR desensitization was 23 ± 7%, while with 5-fold G_iα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of G_i on desensitization was receptor-specific, in that forskolin responses were not altered by G_iα2 overexpression. Thus, acquired β₂AR coupling to G_i is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase G_i levels contribute to β₂AR dysfunction.     

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis

BackgroundIncreasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis.PurposeThe purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis.MethodsA gaussia princeps luciferase–based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and β-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining.ResultsCelastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions.ConclusionTaken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.     

Antagonism of 5-HT1A receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT7 receptors

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5‐HT) receptor subtype next to the 5‐HT_1A autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5‐HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5‐HT₇ receptors. Experiments were performed with the specific 5‐HT₇ antagonist SB 258741 and the putative 5‐HT₇ agonist AS19. In this study WAY 100.635 was used to block 5‐HT_1A receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5‐HT in the ventral hippocampus as well as 5‐HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co‐administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5‐HT in ventral hippocampus, hinting at opposed 5‐HT₇ receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell‐shaped dose–effect curve: moderately increasing 5‐HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5‐HT₇ receptors and moderate affinity for 5‐HT_1A receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5‐HT neuronal activity mediated by 5‐HT₇ receptors. It can be speculated, that the restoration of 5‐HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5‐HT_1A receptors alone, in fact results from a shift in balance between 5‐HT_1A and 5‐HT₇ receptor function.     

Lack of Rapid Desensitization of Ca2+ Responses in Transfected CHO Cells Expressing the Rat Neurotensin Receptor Despite Agonist-Induced Internalization

Abstract: Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca²⁺]_i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca²⁺]_i resulted from release of Ca²⁺ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca²⁺]_i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for ～70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells. A possible absence of desensitization of the neurotensin receptor itself is also discussed.     

Polarity of A2b adenosine receptor expression determines characteristics of receptor desensitization

It is not knownif, in polarized cells, desensitization events can be influenced by thedomain on which the receptor resides. Desensitization was induced by5'-(N-ethylcarboxamido)adenosine (NECA) and wasquantitated by measurement of short-circuit current (I_sc) in response to adenosine. NECA addedto either the apical or basolateral compartments rapidly desensitizedreceptors on these respective domains. Although apical NECA had noeffect on the basolateral receptor stimulation, basolateral NECAinduced a complete desensitization of the apical receptor. Wehypothesized that desensitization of apical receptor by basolateraldesensitization could relate to a trafficking step in which A2breceptor is first targeted basolaterally upon synthesis and transportedto the apical surface via vesicular transport/microtubules. Becausedesensitization is associated with downregulation of receptors, apicaladenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I_scinduced by apical and not basolateral adenosine. In conclusion:1) a single receptor subtype, here modeled by the A2b receptor,differentially desensitizes based on the membrane domain on which it isexpressed, 2) agonist exposure on one domain can result indesensitization of receptors on the opposite domain, 3)cross-domain desensitization can display strict polarity, and4) receptor trafficking may play a role in thecross-desensitization process.

     

Plasma membrane depolarization and activation of receptors for endogenous vasoconstrictors as possible mechanisms of potentiation of vasoconstrictive response to serotonin in traumatic shock in rats

The goal of this work was to study possible mechanisms underlying the potentiation of vasopressor response to serotonin observed in traumatic shock. Experiments with isolated aorta and mesenteric artery of the rat showed that vasoconstriction is caused by the activation of 5HT2A receptors. Agonists of 5HT1B, 5HT1D, 5HT2B, and 5HT4 receptors induced vasodilation. Agonists of 5HT1A receptors had a dual effect determined by interaction with α₁-adrenergic receptors and 5HT1A receptors. Plasma membrane depolarization with 15 mM KCl increased the vasoconstrictive force in response to serotonin. This effect was determined by the ability of KCl to activate voltage-gated calcium channels, as a result of which the intracellular calcium stores are replenished. Inhibition of the response to serotonin by ketanserin, a 5HT2A receptor blocker, did not depend on the presence of 15 mM KCl. Constriction in response to serotonin was potentiated after its addition to vessels preconstricted with noradrenaline or endothelin-1. The constriction response partially retained in the presence of 2 × 10^?7 M ketanserin, which completely suppressed the serotonin-induced constriction of dilated vessels both at normal membrane potential and after plasma membrane depolarization. It can be assumed that noradrenalin and endothelin-1 alter the characteristics of 5HT2A receptors and possibly 5HT1A receptors as a result of their heterodimerization with the receptors for these vasoconstrictive hormones or receptor-receptor interaction at the level of signaling systems. Along with the potentiating effect of KCl, this mechanism may underlie the enhancement of vasopressor response to serotonin in shock.     

Measurement of changes in functional muscarinic acetylcholine receptor density in single neuroblastoma cells using calcium release kinetics

Calcium release in response to the activation of muscarinic M1 and histamine H₁ receptors was studied in single N1E-115 cells using Fura-2 imaging. The objective was to relate changes in the kinetics of Ca release with reductions in functional receptor density resulting from receptor desensitization. Calcium release increased and its time course accelerated with increasing carbachol concentration with an EC₅₀ = 96 ± 8 μM. This value is similar to the binding K_D (100 μM) and the similarity shows that the activation of calcium release is limited by the number of muscarinic receptors. In contrast, the EC₅₀ for Ca release in response to histamine is 4.0 ± 0.7 μM while the binding K_D is 8.3 μM and, therefore, H₁ receptors appear to be in approximately 2-fold excess over the minimum number necessary to fully engage the Ca release mechanism.Functional surface receptor number was assayed in the population of cells by counting the total number of cells responding to agonist. A 5 min exposure to 1 mM carbachol caused 12% of cells to lose their ability to respond to carbachol, with no change in their response to histamine. Interpolating from the dose-response curve taken before desensitization, this is equivalent to an average 23% reduction in the number of muscarinic receptors. In individual cells the latency to Ca release is dose-dependent in the absence of excess receptors. The loss of functional receptors was therefore estimated from the increase in latency after desensitization, and varied from 5–48% of receptors (22 ± 18%). Muscarinic desensitization did not depend on IP₃-evoked Ca release, Ca entry, protein kinase C, NO, or cGMP. We conclude that in a population, the number of cells responding and in single cells, the latency to Ca release can serve as measures of functional receptor density.     

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Purification of HA-tagged P2Y₂ receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y₂ receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y₂ nucleotide receptors from metabolically [³²P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [³²P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y₂ receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y₂ receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y₂ receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y₂ nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005)     

Pharmacological profile of a model for central serotonin receptor activation

The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central cholinergic or GABAergic mechanisms did not alter 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no significant effect on head shakes. However, desmethylimipramine was equipotent with methysergide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&;F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The H₁ receptor antagonist pyrilamine had no effect on head shakes. It is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is particularly sensitive to 5HT antagonists.     

Full and Partial Agoists Induce Distinct Desensitized States of the 5-HT3 Receptor

Abstract

5-HT, receptor-mediated ion currents evoked by the full agonists 5-hydroxy-tryptamine (5-HT), quatemary 5-HT (5-HTQ), meta-chlorophenylbiguanide (mCPBG) and the partial agonists dopamine and tryptamine have been investigated in whole-cell voltage clamp experiments on N1E-115 mouse neuroblastoma cells. All agonists desensitize the 5-HT₃ receptor completely with a steep concentration dependence and a potency order of: mCPBG > 5-HTQ = 5-HT >> tryptamine > dopamine. The time course of recovery from desensitization depends on the agonist used. Recovery from partial agonist-induced desensitization is single exponential. whereas the desensitization induced by full agonists recovers with sigmoid kinetics, suggesting at least 3 transitions between 4 states. It is concluded that full and partial agonists induce distinct desensitized states.     

Variability of calcium responses to agonists of glutamate receptors in hippocampal neurons

The subject of this work was to study the reasons of the variability of the calcium response amplitudes in individual neurons of the hippocampal cell culture to agonists of ionotropic glutamate receptors and the regularities of the calcium response amplitude distribution. Changes of [Ca²⁺] _i in the neurons in response to the NMDA-, AMPA-, and KA-receptor agonists were recorded using fluorescence probe Fura-2. The calcium response amplitudes (expressed as the ratio of fluorescence intensities of Fura-2 upon excitation at wave-lengths 340 and 380 nm) to short-term application of glutamate receptor agonists N-methyl-D-aspartate (NMDA), domoic acid (DA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and (S)-(−)-5-fluorowillardiine (FW) were measured. Calcium responses of individual cells differed in shape and amplitude but always reproduced upon the second application of the agonist. To elucidate the nature of calcium response variability, we compared distributions of calcium response amplitudes to the NMDA-, KA-, and AMPA-receptor agonists in cultures of various ages in the presence of receptor desensitization inhibitors and different agonist concentrations. An even increase from 0.05 to 1.6 was characteristic for distributions of calcium response amplitudes. Nevertheless, in 1–3% neurons of the cell culture, calcium response amplitudes reached much higher values. The efficiency of the ligands usually increased in the following order: FW ≈ NMDA > DA. However, this regularity varied with age and depended on the presence of the receptor desensitization inhibitor. In the process of growth and differentiation of neurons in culture from 1 to 14 day in vitro, calcium response amplitude to AMPA- and KA-receptor agonists increased. Desensitization inhibitors transformed the response from pulse-like with a sharp peak into stepwise and increased the amplitude of calcium responses but did not abolish the character of even amplitude distribution. The effect of AMPA- and KA-receptor desensitization inhibitor decreased with calcium response amplitude growth in the control and approached zero in neurons with initially maximal amplitude. KA- and AMPA-receptor agonists at high concentrations possessed a property of desensitization inhibitors and transformed a transient response into a continuous one that lasted throughout the application time. Thus, the amplitude and shape of the calcium response to glutamate receptor agonists is a characteristic parameter of an individual cell.