Characterization and regulation of insulin binding by embryonic chick heart cells

We have compared insulin binding by heart cells at 7 and 14 days of development. Species specificity, optimum pH, temperature relationships, and time to equilibrium for binding of insulin were the same in both 7 and 14-day systems. Curvilinear Scatchard plots for chicken insulin binding were demonstrated. Binding affinities and capacities were calculated based on a two-receptor model including a specific high-affinity receptor and a less specific low-affinity receptor that bound insulin and other growth peptides. Apparent association constants (K_A) were 4.0 and 0.05 nM^?1 and binding capacities were 600 and 9000 sites per cell for high- and low affinity receptors, respectively. We have also investigated the ability of insulin to regulate binding to its own receptors. Chick heart cells from 7- and 14-day embryos, cultured for 44 hr in insulin-enriched medium (3.4 μM), bound 50% less insulin (down-regulated) than control cells. At both developmental stages, down regulation was primarily a reduction in binding to the high-affinity receptor. The low-affinity receptor was less susceptible to down regulation and retained its ability to mediate maximal insulin stimulation of amino acid transport.     

2- and 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosine receptors

The synthesis of a series of 9-ethyladenine derivatives bearing alkynyl chains in 2- or 8-position was undertaken, based on the observation that replacement of the sugar moiety in adenosine derivatives with alkyl groups led to adenosine receptor antagonists. All the synthesized compounds were tested for their affinity at human and rat A₁, A_2A, and A₃ adenosine receptors in binding assays; the activity at the human A_2B receptor was determined in adenylyl cyclase experiments. Biological data showed that the 2-alkynyl derivatives possess good affinity and are slightly selective for the human A_2A receptor. The same compounds tested on the rat A₁ and A_2A subtypes showed in general lower affinity for both receptors. On the other hand, the affinity of the 8-alkynyl derivatives at the human A₁, A_2A, and A_2B receptors proved to be lower than that of the corresponding 2-alkynyl derivatives. On the contrary, the affinity of the same compounds for the human A₃ receptor was improved, resulting in A₃ selectivity. As in the case of the 2-alkynyl-substituted compounds, the 8-alkynyl derivatives showed decreased affinity for rat receptors. However, it is worthwhile to note that the 8-phenylethynyl-9-ethyladenine was the most active compound of the two series (K_i in the nanomolar range) at both the human and rat A₃ subtype. Docking experiments of the 2- and 8-phenylethynyl-9-ethyladenines, at a rhodopsin-based homology model, gave a rational explanation of the preference of the human A₃ receptor for the 8-substituted compound.     

Synthesis of novel 1-alkyl-8-substituted-3-(3-methoxypropyl) xanthines as putative A2B receptor antagonists

In order to identify a high-affinity, selective antagonist for the A_2B subtype adenosine receptor, more than 40 1,8-disubstituted-3-(3-methoxypropyl) xanthines were prepared and evaluated for their binding affinity at recombinant human adenosine receptors, mainly of the A_2A and A_2B subtypes. Some of the 1-ethyl-3-(3-methoxypropyl)-8-aryl substituted derivatives 15(a–m) showed moderate-to-high affinity at human A_2B receptors, with compound 15d showing A_2B selectivity over the other A receptors assayed (A₁, A_2A, A₃) of 34-fold or over.     

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor

On the basis of a pyrazine core structure, three new adenosine A_2B receptor ligands (7a–c) were synthesized containing a 2-fluoropyridine moiety suitable for ¹⁸F-labeling. Compound 7a was docked into a homology model of the A_2B receptor based on X-ray structures of the related A_2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A₁ receptor, 7a was radiolabeled as the most suitable candidate (K_i(A_2B)?=?4.24?nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [¹⁸F]7a with a standardized uptake value (SUV) of ≈1 at 5?min post injection followed by a fast wash out. Metabolism studies of [¹⁸F]7a in mice revealed the formation of a blood–brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

The Postnatal Development of the Gabaa/benzodiazepine Receptor in the Rat Red Nucleus

Abstract

The development of the GABA_A/Benzodiazepine receptor (GABA_AR) in the red nucleus was studied using ³H-flunitrazepam (FNZ) as the probe. Saturation binding assay showed that the ^Bmax of the ligand to the membranes of the nucleus increased from 0.50 ± 0.04 nmol/mg protein at postnatal day 4, to 0.71 ± 0.1 and 0.78 ± 0.08 at day 7 and day 10. At day 20 the ^Bmax decreased to a level near day 4 and persisted until day 40. However, the affinity of ³H-FNZ to the receptor remained quite constant. At least 4 proteins of 51kD, 53kD, 59kD and 62kD in the nucleus were labeled by ³H-FNZ, as revealed from photoaffinity binding and SDS-PAGE. The labeling of 53kD, 59kD and 62kD was high at earlier ages than day 10, whereas the 51kD was predominent from day 10 to day 40. Receptor binding autoradiography of the nucleus also showed that the most dense labeling was seen around day 10. The early transient increase in the GABA_AR of the red nucleus may indicate the plasticity of the nucleus in response to environmental changes after birth.     

[3H]Ketanserin Binding in Human Brain Postmortem

This study aimed at comparing the binding characteristics of [³H]ketanserin, a high-affinity serotonin 2A (5-HT_2A) receptor antagonist, in the prefrontal cortex, hippocampus and striatum of human brain post-mortem. The results indicated the presence of a single population of binding sites in all the regions investigated, with no statistical difference in maximum binding capacity (B_max) or dissociation constant (K_d) values. The pharmacological profile of [³H]ketanserin binding was consistent with the labeling of the 5-HT_2A receptor, since it revealed a competing drug potency ranking of ketanserin = spiperone > clozapine = haloperidol > methysergide > mesulergine > 5-HT. In conclusion, the 5-HT_2A receptor, as labeled by [³H]ketanserin, would seem to consist of a homogenous population of binding sites and to be equally distributed in human prefronto-cortical, limbic and extrapyramidal structures.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Synthesis and pharmacological evaluation of novel 1,3,8- and 1,3,7,8-substituted xanthines as adenosine receptor antagonists

A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A₁, A_2A, A_2B and A₃ subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A_2B and A₁ receptors, with the most active compound (14q) having a pK_i of 7.57 nM for hA_2B receptors and a selectivity over hA_2A receptors of 8.1-fold and hA₁ receptors of 3.7-fold.     

Bicuculline-insensitive GABA binding to catfish neuronal membranes

GABA receptor binding to mammalian neuronal membranes has been classified into at least 2 subtypes—GABA_A and GABA_B binding sites. In catfish brain GABA_A receptor sites have previously been demonstrated. Evidence is now presented that under appropriate conditions which rule out GABA_A receptor binding, [³H]GABA binds to membranes prepared from catfish brain. This binding is bicuculline-insensitive but differs enough from mammalian GABA_B binding to cast some doubt on the idea that GABA_B receptors exist in catfish brain. Specific binding was detected that was saturable and exhibited a dissociation constant of 4μM. (±)Baclofen, a potent inhibitor in rat brain, was a weak inhibitor, producing a maximum of 43% inhibition. This inhibitory effect could be enhanced, however, in the presence of 320 μM isoguvacine. [³H]GABA binding was unaffected by bicuculline. Thus bicuculline-insensitive GABA binding sites exist in catfish brain but they differ in a number of ways from the GABA_B receptor site found in mammals. Furthermore, a third [³H]GABA binding site appears to exist that is both baclofen- and bicuculline-insensitive, yet is inhibited by high concentrations of isoguvacine, a known GABA_A agonist.     

Oligomeric states of the insulin receptor: binding and autophosphorylation properties

Properties of oligomeric states of the insulin receptor were analyzed by polyacrylamide gel electrophoresis in nondenaturing buffer conditions (ND-PAGE). Partially purified insulin receptors resolve in ND-PAGE as three distinct species: (i) the fast electrophoretic mobility, low molecular mass form manifests intense labeling by iodinated insulin and shows basal and insulin-stimulated autophosphorylation; (ii) the middle, intermediate mobility form exhibits strong labeling by iodinated ligand but does not possess the capacity to be autophosphorylated; (iii) the slow mobility, highest molecular mass form necessitates covalent binding with iodinated hormone to withstand electrophoresis and shows autophosphorylation enhanced by insulin. This receptor form is more heavily labeled by phosphorylation than the low form. At 22 degrees C, binding and autophosphorylation do not appear to be time dependent. At 37 degrees C, binding and autophosphorylation of low and high species attain a maximum after 15 min and then decrease as time of incubation with insulin is prolonged to 120 min; the middle species exhibits a much slower association rate, and its labeling by iodinated hormone becomes more intense with time. Our data show that in cell-free systems insulin receptors appear in various oligomeric states and that the highest molecular mass oligomer exhibits the most pronounced autophosphorylation. This is compatible with the concept that insulin receptor oligomerization provides a mechanism for transmembrane signaling.     

Novel human adenosine receptor antagonists based on the 7-amino-thiazolo[5,4-d]pyrimidine scaffold. Structural investigations at the 2-, 5- and 7-positions to enhance affinity and tune selectivity

This paper describes the synthesis of novel 7-amino-thiazolo[5,4-d]pyrimidines bearing different substituents at positions 2, 5 and 7 of the thiazolopyrimidine scaffold. The synthesized compounds 2–27 were evaluated in radioligand binding (A₁, A_2A and A₃) and adenylyl cyclase activity (A_2B and A_2A) assays, in order to evaluate their affinity and potency at human adenosine receptor subtypes. The current study allowed us to support that affinity and selectivity of 7-amino-thiazolo[5,4-d]pyrimidine derivatives towards the adenosine receptor subtypes can be modulated by the nature of the groups attached at positions 2, 5 and 7 of the bicyclic scaffold. To rationalize the hypothetical binding mode of the newly synthesized compounds, we also performed docking calculations in human A_2A, A₁ and A₃ structures.     

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A₁, A_2A and A₃ adenosine receptors and in adenylyl cyclase experiments for their potency at the human A_2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N ⁶ position of 9-propyladenine significantly increased binding affinity at the human A₁ and A₃ adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N ⁶ position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A_2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A_2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Alterations in hippocampal serotonergic and INSR function in streptozotocin induced diabetic rats exposed to stress: neuroprotective role of pyridoxine and <Emphasis Type="Italic">Aegle marmelose</Emphasis>

Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin, Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT_2A receptor subtype, gene expression studies on 5-HT_2A, 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters B_max and K_d showed a significant decrease (p < 0.001) whereas 5-HT_2A receptor binding parameters B_max showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in K_d in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT_2A, 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, B_max , K_d of 5-HT, 5-HT_2A and gene expression of 5-HT_2A, 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT_2A and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.     

Synthesis and pharmacological evaluation of novel 1- and 8-substituted-3-furfuryl xanthines as adenosine receptor antagonists

The synthesis of an important set of 3-furfurylxanthine derivatives is described. Binding affinities were determined for rat A₁ and human A_2A, A_2B and A₃ receptors. Several of the 3-furfuryl-7-methylxanthine derivatives showed moderate-to-high affinity at human A_2B receptors, the most active compound (10d) having a K_i of 7.4 nM for hA_2B receptors, with selectivities over rA₁ and hA_2A receptors up to 14-fold and 11-fold, respectively. Affinities for hA₃ receptors were very low for all members of the set.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

Molecular modelling study of 2-phenylethynyladenosine (PEAdo) derivatives as highly selective A3 adenosine receptor ligands

A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N⁶- and 4′-position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A₁, A_2A, A₃) or adenylyl cyclase activity assay (A_2B). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A₃ receptors, resulting in compounds endowed with high affinity and selectivity for the A₃ subtype. Additional substitution of the N⁶- and 4′-position increases both A₃ affinity and selectivity. The results showed that the new compounds have a good affinity for the A₃ receptor and in particular, the N⁶-methoxy-2-phenylethynyl-5′-N-methylcarboxamidoadenosine, with a K_i at A₃ of 1.9 nM and a selectivity A₁/A₃ and A_2A/A₃ of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A₃ adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A₃ receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A₃ receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data.An erratum to this article can be found at     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.