Lack of a difference between ketanserin and ritanserin in central vs. peripheral serotonin receptor antagonism

Ketanserin and ritanserin antagonized with similar potency the pressor response to serotonin in pithed rats, a measure of antagonism of vascular 5HT2 receptors. Both compounds also antagonized the elevation of serum corticosterone concentration by quipazine, a centrally acting serotonin agonist; higher doses of both antagonists were needed, but ketanserin was not less potent than ritanserin. Earlier suggestions that ketanserin mainly blocks peripheral serotonin receptors and that ritanserin mainly blocks central serotonin receptors seem unfounded.     

The role of the 5-HT2 receptor in the regulation of sexual performance of male rats

The present studies have attempted to evaluate the role of 5-HT2 receptors in the regulation of sexual behavior of male rats by determining the effects of 5-HT2 receptor antagonists, pirenperone, LY53857 and LY281067, and a 5-HT2 receptor agonist, DOI. The administration of 1 mg/kg s.c. pirenperone produced a total suppression of ejaculatory response and lower doses had no effect. However, the administration 0.1 mg/kg s.c. of either LY53857 or LY281067 restored ejaculatory capacity to rats that were unable to ejaculate and produced significant decreases in ejaculatory latency in rats with full sexual capacity. Although all of these agents are 5-HT2 antagonists, LY53857 and LY281067 lack the additional monoaminergic activity of pirenperone. Since the effects of pirenperone were opposite from the effects of the selective 5-HT2 antagonists, the suppressive effects of this agent were probably related to its other monoaminergic activity e.g. alpha 1 antagonist activity. This proposal was supported by the observation that the administration of prazosin, an alpha 1 antagonist, significantly increased ejaculatory latency and suppressed the stimulatory effects of LY53857. In contrast to the stimulatory effects of the selective 5-HT2 antagonists, the administration of DOI, resulted in a suppression of sexual performance, which was blocked by pretreatment with LY53857.     

Thyroidectomy blocks stress-induced prolactin rise in rats: role of the central serotonergic system.

Thyroidectomized (TX) adult Wistar male rats and their sham-operated controls were submitted to immobilization stress during forty minutes. Thyroidectomy partially blocks stress-induced prolactin (PRL) secretion. Previous administration of MK 212, a serotonin agonist, reverts this picture. The effect of MK 212 is specifically due to its interaction with 5HT2 receptors, since the injection of LY 53857, a selective blocker of these receptors, 30 min before MK 212, prevents the effect of this serotonin agonist. LY 53857, injected alone, yields a partial blockade of PRL secretion during stress in sham-operated rats. TX rats receiving LY 53857 or saline have comparable low values of plasma PRL during stress. It is suggested that thyroidectomy disrupts the functional integrity of the central serotonergic pathways involved in the stress-induced PRL rise.     

Effect of LY53857, a selective 5HT2 receptor antagonist, on 5HT-induced increases in cutaneous vascular permeability in rats

Both serotonin and histamine increased cutaneous vascular permeability in rats; however, serotonin was approximately 100-fold more potent than histamine. LY53857 (0.1 and 1.0 mg/kg, i.p.), a selective 5HT2 receptor antagonist, blocked serotonin- but not histamine-induced increases in cutaneous vascular permeability. the alpha 1 receptor antagonist, prazosin, did not significantly affect increases in vascular permeability produced by serotonin. These data extend previous studies with LY53857 by further documenting its selectivity as a 5HT2 receptor antagonist. In addition, these results with a selective 5HT2 receptor antagonist provide evidence that 5HT2 receptor activation may be the predominant mechanism associated with vascular permeability changes induced by serotonin.     

A new interpretation of the involvement of serotonin in delayed-type hypersensitivity. Serotonin-2 receptor antagonists inhibit contact sensitivity by an effect on T cells

5-HT is a neuromediator and a vasoactive amine released by platelets and murine mast cells at sites of inflammation. A role for 5-HT has been proposed in murine DTH and has been attributed to its 5-HT2R-dependent vasoactive properties. We have tested the hypothesis that the role of 5-HT in DTH is related to an interaction of 5-HT with DTH effector T cells. In vivo treatment of sensitized mice with the 5-HT2R antagonists methysergide or ketanserin inhibited both their capacity to elicit DTH and the ability of their lymphoid cells to transfer DTH. In vitro treatment of lymphoid cells, or of nylon wool-purified T cells from sensitized mice, with 10(-7) to 10(-9) M of the 5-HT2R antagonists methysergide, ketanserin, ritanserin, or LY 53857, followed by three washings, inhibited as strongly their ability to transfer DTH, both systemically or locally. Systemic and local co-transfer experiments of 5-HT2R antagonist-treated and untreated cells indicated that this inhibition was not related to the induction of suppression. 5-HT2R antagonist treatment was nontoxic to T cells; did not affect the in vitro response of T cells to mitogen; selectively inhibited the efferent, but not the afferent limb of DTH; and in the efferent T cell cascade, affected the late-acting (24 h) inflammatory DTH T cells, but not the early-acting, DTH-initiating T cells. 5-HT2R selectivity was suggested by the absence of effect of an alpha-adrenergic R antagonist, and by prevention of the inhibitory effect of a 5-HT2R antagonist by prior incubation with the selective 5-HT2R agonist 1-(2,5-dimethoxy phenyl-4-methyl)-2 aminopropane. In summary, inhibition of DTH effector T cell function appeared sufficient, independently of any vascular effect, to account for the in vivo inhibitory effect of 5-HT2R antagonists on the elicitation of DTH. Our data suggest that late-acting DTH effector T cells might express functional 5-HT2R, and that these receptors might require in vivo activation in order for the T cells to locally produce the inflammatory lymphokine-dependent aspects of DTH.     

5-hydroxytryptamine and precapillary vessels

The complexity of the vascular effects of 5-hydroxytryptamine (5-HT) is illustrated by differences in sensitivity to the amine among arterial tissues of different origin. The interaction of 5-HT with 5-HT2 receptors is inhibited by specific antagonists such as ketanserin and methysergide. Such compounds also inhibit the contractile responses to endogenous 5-HT released from aggregating platelets. The vasodilator component of the response to 5-HT can be unmasked in the presence of serotonergic blockade, provided the antagonist used has no partial agonistic properties. 5-HT augments (amplifies) the vasoconstrictor responses to adrenergic and nonadrenergic neurohumoral mediators. The amplifying effect of the monoamine is prevented by 5-HT2-serotonergic antagonists such as ketanserin.     

The serotonin 5-HT2 receptor system, but not the alpha 1-adrenergic receptor system, is involved in the estrogen-induced afternoon prolactin surge in the rat

Ketanserin (Ket), a new serotonergic (5-HT2) antagonist, has recently been shown to block the estrogen-induced afternoon PRL surge (Endocrinology 120: 2070-2077, 1987). It is not certain, however, whether the effect of Ket was due to its serotonergic or adrenergic receptor antagonistic property. Another 5-HT2 receptor antagonist, LY53857, which possesses no alpha 1-adrenergic receptor affinity, as well as an alpha 1-adrenergic receptor antagonist, prazosin, were used in this study to further clarify the mechanism of 5-HT in the control of PRL secretion. Adult female Sprague-Dawley rats ovariectomized for 2-3 weeks and given a single injection of polyestradiol phosphate were studied 6 days later. Ket, LY53857 and prazosin were examined singly or in combination and animals were injected twice on the sampling day at 1200 and 1300h, respectively. The dosages were as follows: Ket and LY53857, 3 mg/kg BW, ip and 2 mg/kg BW, sc; prazosin, 1 mg/kg BW, ip and 0.7 mg/kg BW, sc. Blood samples were drawn from indwelling intraatrial catheters throughout the afternoon PRL surge.     

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Response suppression induced with selective 5-HT agonists can be differentially blocked with LY53857 in an animal model of depression

Studies from this laboratory have demonstrated that administration of the selective 5-HT_2/1C antagonist LY53857 can block 5-HTP-induced response suppression. To further investigate the serotonergic mechanisms involved in this effect, we decided to test the capacity of LY53857 to block response suppression induced with two selective 5-HT agonists. After a 15 minute baseline period, rats trained to press a lever for milk reinforcement on a VI 1 schedule were given IP injections of 1.0 mg/kg DOI, or 1.0 mg/kg 8-OH-DPAT to induce response suppression. Subsequently, rats were injected with 1.0 mg/kg LY53857 1 hour prior to DOI- or 8-OH-DPAT-induced response suppression. Preinjections with LY53857 resulted in a 100% blockade of DOI-induced response suppression whereas the same dose did not block response suppression induced with 8-OH-DPAT. These results indicate that the 5-HTP-induced response suppression shows some pharmacological similarity to DOI-induced response suppression and may be mediated through 5-HT₂ and/or 5-HT_1C receptors.Special issue dedicated to Dr. Morris H. Aprison     

Norepinephrine-mediated Regulation of 5HT<Subscript>1</Subscript> Receptor Functioning in Human Platelets

Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.     

Antihypertensive properties of ketanserin (R 41 468)

The serotonergic receptor antagonist 3-(2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl)-2,4-[1H, 3H] quinazolinedione (ketanserin) causes dose-dependent inhibition of the effects of 5-hydroxytryptamine (5-HT) on 5-HT2-serotonergic receptors. These receptors mediate facilitation of platelet aggregation, direct vasoconstriction in several arteries and veins, and direct amplification of vasoconstrictor responses to other neurohumoral mediators. Ketanserin does not inhibit vasodilator effects of 5-HT. At higher concentrations, ketanserin has alpha 1-adrenergic blocking properties. The compound causes dose-related reductions in arterial blood pressure in hypertensive animals and humans that are larger and occur at lower doses than in normotensive controls. In humans, the antihypertensive properties of ketanserin do not appear to involve alpha 1-adrenergic inhibition, because the compounds given i.v. (10 mg) do not affect the pressor dose-response curve to phenylephrine.     

Shape change and uptake of 5-hydroxytryptamine in human blood platelets: action of neuropsychotropic drugs.

Psychotropic drugs acted as agonists, antagonists or mixed agonists-antagonists of the shape change mediated by the 5HT receptor in blood platelets of man incubated in a protein-poor medium. Regarding inhibition of 5HT uptake some of the drugs were equally potent in both reserpinized and normal platelets, whereas others showed lower or higher potencies in the former than in the latter. There was no correlation between the potencies of the drugs as shape change inhibitors and as uptake inhibitors. It is concluded that 1) human platelets may serve as models for the neuronal 5HT receptors of some areas of the central nervous system, 2) platelets can be used to determine the subcellular site of action of 5HT uptake inhibitors and 3) the receptors responsible of the shape change, and the site of the 5HT transport at the plasma membrane are different entities.     

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.     

5HT2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition

Contractile responses to serotonin were examined $\text{in}$$\text{vitro}$ in the longitudinal portal vein to determine whether such responses were mediated by the interaction of serotonin with 5HT₁ receptors (those that preferentially bind [³H]serotonin) or 5HT₂ receptors (those that preferentially bind [³H]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the affinity for serotonin receptors in the rat portal vein and the ability to bind to 5HT₂, but no 5HT₁ receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED₅₀ value) relative to contractions produced by the non-cumulative addition of serotonin. Affinities of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentration-response curves compared to affinities obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT₂ receptor affinities of antagonists in the rat portal vein are best determined by the shift of non-cumulative responses to serotonin.     

Effects of serotonergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami

Experimental evidence suggests that serotonin (5HT) is excitatory to the hypothalamic-pituitary-adrenal axis and that this effect involves activation of both hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion. The present study was undertaken to examine the mechanism by which 5HT stimulates the central component of the HPA axis. To accomplish this we employed an in vitro rat hypothalamic organ culture system in which CRH secretion from single explanted hypothalami was measured by specific radioimmunoassay (IR-rCRH). All experiments were performed after an overnight (15-18 hr) preincubation. Serotonin stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped and the peak effect was observed at the concentration of 10(-9) M. The stimulatory effect of 10(-9) M 5HT was antagonized by the 5HT1 and 5HT2 receptor metergoline and by the selective 5HT2 receptor antagonists ketanserin and ritanserin. The muscarinic antagonist atropine, the nicotinic antagonist hexamethonium and the alpha-adrenergic receptor antagonist phentolamine, on the other hand, did not inhibit 5HT-induced IR-rCRH secretion. The specific 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped with peak of effect reached at the concentration of 10(-9) M. We also tested the ability of the 5HT agonist meta-chlorophenylpiperazine (m-CPP) and of the selective 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to cause CRH secretion. Although both m-CPP and 8-OH-DPAT stimulated IR-rCRH secretion in a dose-dependent fashion, several differences were observed when their effect was compared to that of 5HT. These included a different shape of the dose-response curve, a lower maximal stimulatory effect and a different maximal stimulatory concentration. These findings suggest that serotonin stimulates CRH secretion by explanted rat hypothalami and that this effect appears to be mediated mainly through a 5HT2 receptor mechanism.     

Serotonin 5-HT2 receptor binding on blood platelets--a peripheral marker for depression?

Several methods of platelet membrane preparation and binding conditions were screened in order to optimize the labeling of serotonergic 5-HT2 receptors on previously frozen human platelet membranes with tritiated ketanserin. Under optimal conditions, 5-HT2 receptors in normal subjects (5 males, 7 females, age range 21 to 71) have a Kd of 1.5 +/- 0.2 nM and a Bmax of 33.9 +/- 5.3 fmole/mg protein. In a group of patients with major depressive disorder exactly matched for age and sex with the normal control group, we find a significant increase in receptor density, to 66.8 +/- 11.4 fmole/mg, with no significant change in the affinity (2.3 +/- 0.5 nM). Four weeks of treatment with antidepressant drugs result in a significant decrease of Bmax, down to control levels (29.4 +/- 3.9). Thus, ketanserin can be used to monitor changes in platelet serotonin 5-HT2 receptors which may be a relevant marker for the state of depression.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Discovery and initial structure-activity relationships of trisubstituted ureas as thrombin receptor (PAR-1) antagonists

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.