The atypical antipsychotics clozapine and olanzapine promote down-regulation and display functional selectivity at human 5-HT7 receptors.

BACKGROUND AND PURPOSE: Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs -coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors. EXPERIMENTAL APPROACH: HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of ß-arrestins and lysosomal trafficking were measured. KEY RESULTS: The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of ß-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants. CONCLUSIONS AND IMPLICATIONS: Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor.

The functional activity of inhibitory G protein (G(i)) is not increased in failing heart ventricle.

Beta-adrenergic receptor (ßAR) inotropic effects are attenuated and muscarinic receptor-mediated inhibition thereof is enhanced in heart failure. We investigated if increased G(i) activity contributes to attenuated ßAR-inotropic effects and potentiates muscarinic accentuated antagonism in failing rat ventricle. Contractility was measured in ventricular strips and adenylyl cyclase (AC) activity in ventricular membranes from rats with post-infarction heart failure (HF) or Sham-operated controls (Sham). The maximal ßAR-mediated inotropic effect of isoproterenol was reduced by ~70% and basal, ßAR- & forskolin-stimulated AC activity was significantly lower in HF vs. Sham. Carbachol-evoked antagonism of the ßAR-mediated inotropic response was complete only in HF despite a ~40% reduction in the ability of carbachol to inhibit ßAR-stimulated AC. However, neither the relative efficacy (contractility decreased by ~46%) nor the potency of carbachol to inhibit the ßAR inotropic response differed between Sham and HF ventricle. Pertussis toxin (PTX) inactivation of G(i) did not increase the maximal ßAR inotropic effect or the attenuated basal, ßAR- & forskolin-stimulated AC activity in HF, but increased the potency of isoproterenol only in Sham (~0.5 log unit). In HF ventricle pretreated with PTX, simultaneous inhibition of phosphodiesterases 3,4 (PDE3,4) alone produced a larger inotropic response than isoproterenol in ventricle untreated with PTX (84% and 48% above basal respectively). In the absence of PTX, PDE3,4 inhibition evoked negligible inotropic effects in HF. These data are not consistent with the hypothesis that increased G(i) activity contributes to the reduced ßAR-mediated inotropic response and AC activity in failing ventricle. The data, however, support the hypothesis that G(i), through chronic receptor independent inhibition of AC, together with PDE3,4 activity, is necessary to maintain a low basal level of contractility.

Cyclic AMP-dependent inotropic effects are differentially regulated by muscarinic G(i)-dependent constitutive inhibition of adenylyl cyclase in failing rat ventricle.

BACKGROUND AND PURPOSE: ß-Adrenoceptor (ß-AR)-mediated inotropic effects are attenuated and G(i) proteins are up-regulated in heart failure (HF). Muscarinic receptors constitutively inhibit cAMP formation in normal rat cardiomyocytes. We determined whether constitutive activity of muscarinic receptors to inhibit adenylyl cyclase (AC) increases in HF and if so, whether it modifies the reduced ß-AR- or emergent 5-HT4-mediated cAMP-dependent inotropic effects. EXPERIMENTAL APPROACH: Contractility and AC activity were measured and related to each other in rat ventricle with post-infarction HF and sham-operated (Sham) controls with or without blockade of muscarinic receptors by atropine and inactivation of G(i) protein by pertussis toxin (PTX). KEY RESULTS: Isoprenaline-mediated inotropic effects were attenuated and basal, isoprenaline- and forskolin-stimulated AC activity was reduced in HF compared with Sham. Atropine or PTX pretreatment increased forskolin-stimulated AC activity in HF hearts. ß-AR-stimulated AC and maximal inotropic response were unaffected by atropine in Sham and HF. In HF, the potency of serotonin (5-HT) to evoke an inotropic response was increased in the presence of atropine with no change in the maximal inotropic response. Interestingly, PTX pretreatment reduced the potency of 5-HT to evoke inotropic responses while increasing the maximal inotropic response. CONCLUSIONS AND IMPLICATIONS: Although muscarinic constitutive inhibition of AC is increased in HF, it does not contribute to the reduced ß-AR-mediated inotropic effects in rat ventricle in HF. The data support the hypothesis that there are differences in the functional compartmentation of 5-HT4 and ß-AR AC signalling in myocardium during HF.

Activation of muscarinic receptors elicits inotropic responses in ventricular muscle from rats with heart failure through myosin light chain phosphorylation.

BACKGROUND AND PURPOSE: Muscarinic stimulation increases myofilament Ca(2+) sensitivity with no apparent inotropic response in normal rat myocardium. Increased myofilament Ca(2+) sensitivity is a molecular mechanism promoting increased contractility in failing cardiac tissue. Thus, muscarinic receptor activation could elicit inotropic responses in ventricular myocardium from rats with heart failure, through increasing phosphorylation of myosin light chain (MLC). EXPERIMENTAL APPROACH: Contractile force was measured in left ventricular papillary muscles from male Wistar rats, 6 weeks after left coronary artery ligation or sham surgery. Muscles were also frozen, and MLC-2 phosphorylation level was quantified. KEY RESULTS: Carbachol (10 micromol.L(-1)) evoked a positive inotropic response only in muscles from rats with heart failure approximating 36% of that elicited by 1 micromol.L(-1) isoproterenol (20 +/- 1.5% and 56 +/- 6.1% above basal respectively). Carbachol-evoked inotropic responses did not correlate with infarction size but did correlate with increased left ventricular end diastolic pressure, heart weight/body weight ratio and lung weight, primary indicators of the severity of heart failure. Only muscarinic receptor antagonists selective for M(2) receptors antagonized carbachol-mediated inotropic effects with the expected potency. Carbachol-evoked inotropic responses and increase in phosphorylated MLC-2 were attenuated by MLC kinase (ML-9) and Rho-kinase inhibition (Y-27632), and inotropic responses were abolished by Pertussis toxin pretreatment. CONCLUSION AND IMPLICATIONS: In failing ventricular muscle, muscarinic receptor activation, most likely via M(2) receptors, provides inotropic support by increasing MLC phosphorylation and consequently, myofilament Ca(2+) sensitivity. Enhancement of myofilament Ca(2+) sensitivity, representing a less energy-demanding mechanism of inotropic support may be particularly advantageous in failing hearts.

Effects of treatment with a 5-HT4 receptor antagonist in heart failure.

BACKGROUND AND PURPOSE: Positive inotropic responses (PIR) to 5-hydroxytryptamine (5-HT) are induced in the left ventricle (LV) in rats with congestive heart failure (CHF); this is associated with upregulation of the G(s)-coupled 5-HT(4) receptor. We investigated whether chronic 5-HT(4) receptor blockade improved cardiac function in CHF rats. EXPERIMENTAL APPROACH: Rats were given either the 5-HT(4) antagonist SB207266 (0.5 mg kg(-1) 24h(-1); MI(int)) or placebo (MI(pl)) through mini-osmotic pumps for 6 weeks subsequent to induction of post-infarction CHF. In vivo cardiac function and ex vivo responses to isoprenaline or 5-HT were evaluated using echocardiography and isolated LV papillary muscles, respectively. mRNA levels were investigated using real-time quantitative RT-PCR. KEY RESULTS: LV diastolic function improved, with 4.6% lower LV diastolic diameter and 24.2% lower mitral flow deceleration in MI(int) compared to MI(pl). SB207266 reduced LV systolic diameter by 6.1%, heart weight by 10.2% and lung weight by 13.1%. The changes in posterior wall thickening and shortening velocity, cardiac output, LV systolic pressure and (dP/dt)(max), parameters of LV systolic function, did not reach statistical significance. The PIR to isoprenaline (10 microM) increased by 36% and the response to 5-HT (10 microM) decreased by 57% in MI(int) compared to MI(pl). mRNA levels for ANP, 5-HT(4(b)) and 5-HT(2A) receptors, MHCbeta, and the MHCbeta/MHCalpha -ratio were not significantly changed in MI(int) compared to MI(pl). CONCLUSIONS AND IMPLICATIONS: Treatment with SB207266 to some extent improved in vivo cardiac function and ex vivo myocardial function, suggesting a possible beneficial effect of treatment with a 5-HT(4) receptor antagonist in CHF.

The cloned human 5-HT7 receptor splice variants: a comparative characterization of their pharmacology, function and distribution.

Serotonin (5-hydroxytryptamine, 5-HT) receptor pre-mRNA is alternatively spliced in human tissue to produce three splice variants, h5-HT7(a), h5-HT7(b) and h5-HT7(d), which differ only in their carboxyl terminal tails. Using membranes from transiently and stably transfected HEK293 cells expressing the three recombinant h5-HT7 splice variants we compared their pharmacological profiles and ability to activate adenylyl cyclase. Using PCR on cDNA derived from various human tissues, the 5-HT7(a) and 5-HT7(b) splice variants were detected in every tissue examined. The h5-HT7(d) splice variant was detected in 13 of 16 tissues examined, with predominant expression in the heart, small intestine, colon, ovary and testis. All three h5-HT7 splice variants displayed high affinity binding for [3H]5-HT (pKd=8.8-8.9) in the presence and absence of 100 microM GTP and had similar binding affinities for all 17 ligands evaluated. In HEK293 cells expressing similar, high levels of receptor (approximately 10,000 fmol/mg protein), 5-CT (5-carboxamidotryptamine), 5-MeOT (5-methoxytryptamine) and 5-HT were full agonists while 8-OH-DPAT ((2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin) was a partial agonist with relative efficacy of approximately 0.8. Even at this high receptor level, EC50 values for stimulation of adenylyl cyclase were 10- to 50-fold higher than the Kd values, indicating a lack of spare receptors. No significant differences in coupling to adenylyl cyclase were observed between the three splice variants over a wide range of receptor expression levels. For antagonists, binding affinities determined by displacement of [3H]5-HT binding and by competitive inhibition of 5-HT-stimulated adenylyl cyclase activity were essentially identical amongst the splice variants. These studies indicate that the three human splice variants are pharmacologically indistinguishable and that modifications of the carboxyl tail do not influence coupling to adenylyl cyclase.

5HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle.

5-Hydroxytryptamine (5-HT) increases human heart rate and atrial contractile force and hastens atrial relaxation through 5-HT4 receptors. Moreover, 5-HT may be arrhythmogenic and give rise to atrial fibrillation. It is not clear which splice variant(s) of the 5-HT4 receptor is expressed and mediates these effects of 5-HT in the human heart. Previous studies have indicated different pharmacological properties of 5-HT4 receptors in human heart and mouse colliculi neurones, possibly due to expression of different splice variants. We therefore cloned the human 5-HT4(b) receptor and compared its pharmacological properties with those of the cloned human 5-HT4(a) receptor and searched for the corresponding mRNA in human tissues. The primary structures of the two human 5-HT4 receptor splice variants are identical except for divergent C-terminal tails of 28 and 29 amino acids in the 5-HT4(a) and 5-HT4(b) receptors, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that both variants were coexpressed in various tissues, including cardiac atrium and ventricle. Additional bands suggested the presence of more than two human 5-HT4 receptor splice variants. With cloned receptors stably expressed in HEK293 cells or transiently expressed in COS-7 cells, [3H]GR 113808 consistently showed slightly higher binding affinity to h5-HT4(b) than to h5-HT4(a) receptor (pKd 0.1-0.2 log units higher). Competition of agonists, partial agonists and antagonists for [3H]GR113808 binding revealed no significant differences between the two receptors. For 5-HT4 receptor agonists and antagonists, their potencies in stimulating or inhibiting, respectively, 5-HT-stimulated adenylyl cyclase activity correlated well with their binding affinities. Tropisetron and SB207710 showed partial agonist activity at high receptor expression levels for both isoforms. Cisapride and renzapride were both partial agonists at moderate receptor levels and full agonists at high receptor levels. Cisapride was more potent than renzapride while the converse was the case in human atrium, for which cisapride had lower affinity and agonist potency than at the recombinant receptors. The binding affinities and agonist potencies of ligands for both 5-HT4(a) and 5-HT4(b) receptors correlated with the corresponding affinities and potencies in human atrium. The agonist potency of SB207710 was around 10 times lower than its binding and blocking affinity for both splice variants, suggesting that activation of adenylyl cyclase and blockade of 5-HT responses are mediated through different conformational states. The similar pharmacological properties of the two human 5-HT4 receptor splice variants together with their expression in human atrium would be consistent with mediation of the cardiostimulant effects of 5-HT through both variants. However, the effects of cisapride appear either mediated through non-a and non-b splice variants of the 5-HT4 receptor or 5-HT4(a) and 5-HT4(b) receptor expression in human atrial cells alters somewhat their pharmacological profile through still unknown mechanisms.

Impaired spatial learning after saturation of long-term potentiation.

If information is stored as activity-driven increases in synaptic weights in the hippocampal formation, saturation of hippocampal long-term potentiation (LTP) should impair learning. Here, rats in which one hippocampus had been lesioned were implanted with a multielectrode stimulating array across and into the angular bundle afferent to the other hippocampus. Repeated cross-bundle tetanization caused cumulative potentiation. Residual synaptic plasticity was assessed by tetanizing a naïve test electrode in the center of the bundle. Spatial learning was disrupted in animals with no residual LTP (<10 percent) but not in animals that were capable of further potentiation. Thus, saturation of hippocampal LTP impairs spatial learning.

Spontaneous and amphetamine-evoked release of cerebellar noradrenaline after sensorimotor cortex contusion: an in vivo microdialysis study in the awake rat.

Microdialysis sampling combined with HPLC was used to assess spontaneous and d-amphetamine (AMPH)-evoked release of noradrenaline (NA) in the cerebellum 1 day after probe implantation and 1 day after contusion of the right sensorimotor cortex (SMCX) in rats. In normal controls the mean +/- SEM basal NA release was 10.08 +/- 0.97 pg in the left cerebellar hemisphere and 8.21 +/- 1.17 pg in the right hemisphere 22-24 h after probe implantation. The average +/- SEM NA release in a 3-h period after administration of AMPH (2 mg/kg, i.p.) increased to 453 +/- 47.35 pg in the left and to 402 +/- 49.95 pg in the right cerebellar hemisphere. NA release (range of 413-951% increase over baseline) was maximal 20-40 min postdrug, returned to basal levels within 5 h, and remained unchanged for the 22-24-h postdrug measurement period. Animals with a focal SMCX contusion had a marked depression of both spontaneous and AMPH-evoked NA release. Mean +/- SEM basal NA release was 4.84 +/- 1.09 pg in the left and 4.95 +/- 0.43 pg in the right cerebellar hemisphere from 22 to 24 h postinjury, with NA levels increasing to 259 +/- 75.44 and 219 +/- 23.45 pg in the respective hemispheres over a 3-h period after AMPH. The maximal AMPH-induced increase in NA release ranged from 522 to 1,088% of basal levels in contused rats, with NA release returning to predrug levels within 5 h and remaining depressed for at least 48 h postinjury.(ABSTRACT TRUNCATED AT 250 WORDS)

Unilateral locus coeruleus lesions facilitate motor recovery from cortical injury through supersensitivity mechanisms.

Previous research has indicated that noradrenergic infusions into the cerebellum contralateral to a sensorimotor cortex injury facilitate recovery of motor function. In the present study, the locus coeruleus was lesioned at 2 weeks prior to, 1 week prior to, or simultaneous with a right sensorimotor cortex injury, and functional recovery in response to noradrenergic cerebellar infusions was measured using the beam-walk task. When the locus coeruleus lesion was separated from the sensorimotor cortex lesion by 1 week or more, noradrenergic-induced facilitation of functional recovery occurred with the greater effects observed at the 2-week interval. Simultaneous locus coeruleus and sensorimotor cortex injury with cerebellar noradrenergic infusions revealed no difference in functional recovery. The results suggest that denervation supersensitivity and/or sprouting developed in the cerebellum following the locus coeruleus lesions if a sufficient amount of time elapsed before the sensorimotor cortex injury. The heightened sensitivity to noradrenergic infusions in the contralateral cerebellum suggests that noradrenergic changes in this structure underlie the acceleration of functional recovery from the cortical injury.

Reinstatement of motor deficits in recovered brain-injured animals: the role of cerebellar norepinephrine.

Previous research has indicated that antagonists of locus ceruleus functioning, when administered during the acute phase of an injury, slow recovery of motor function following unilateral sensorimotor cortex injury. Following a recovery plateau in animals, it is possible to pharmacologically reinstate unilateral motor deficits in recovered animals with similar acting drugs given intraperitoneally. The present study was designed to localize the brain systems responsible for the reinstatement of the deficit after recovery from the cortical injury. The results indicate that maintaining functional recovery after injury is modulated by NE in the cerebellum contralateral to the injury, since microinfusions of phenoxybenzamine into this structure reinstate motor deficits. Additionally, removal of the noradrenergic projection to contralateral cerebellum through unilateral lesions of the locus ceruleus reinstate unilateral deficits more severely than the drug administration.

Unilateral, but not bilateral, locus coeruleus lesions facilitate recovery from sensorimotor cortex injury.

This study investigates the role of the locus coeruleus in recovery from sensorimotor cortex injury. Unilateral locus coeruleus lesions given 2 weeks prior to unilateral sensorimotor cortex injury facilitate subsequent motor recovery compared to animals with only a sensorimotor cortex injury, while bilateral locus coeruleus lesions severely retard motor recovery. The results suggest that recovery of function from the cortical injury is facilitated as long as a sufficient amount of the noradrenergic system remains intact, perhaps to provide a basis for compensatory sprouting. The results also suggest that recovery does occur in the absence of the locus coeruleus, indicating that the noradrenergic system is not necessary for recovery to occur after the cortical injury.

Cerebellar norepinephrine infusions facilitate recovery after sensorimotor cortex injury.

This study reports the effects of norepinephrine infusions into cerebellum after unilateral sensorimotor cortex injury. The results demonstrate an immediate and permanent acceleration in motor recovery in awake rats infused with 150 micrograms norepinephrine into the cerebellum contralateral to a right sensorimotor cortex ablation. A vehicle infusion or infusion of norepinephrine into the ipsilateral cerebellum produced no beneficial effects on functional recovery.