Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells

The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Contraction, relaxation, inositol 1,4,5-triphosphate (IP₃) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. 5-HT binding was completely inhibited by 5-HT and partially by 5-HT_2A (ketanserin), 5-HT₄ (SDZ-205,557), and 5-HT_1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP₃, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT_2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT_2A receptors mediating contraction and 5-HT₄ receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT₄ receptors are closely similar or identical to 5-HT_1p receptors.     

Duration of Action of Inhaled vs. Intravenous β2-Adrenoceptor Agonists in an Anaesthetized Guinea-pig Model

We compared the duration of action of the short-acting α₂-adrenoceptor agonist salbutamol and the long-acting α₂-adrenoceptor agonists salmeterol and formoterol when administered iv or by inhalation in a histamine-induced bronchoconstriction model in the guinea-pig. Following aerosol dosing, maximal bronchoprotector effects were seen for salbutamol, salmeterol and formoterol at concentrations of 1 mg/ml, 100 μ g/ml and 30 μ g/ml respectively, giving a potency order of formoterol > salmeterol > salbutamol. All displayed similar maximum effects in this system. A maximal concentration of salbutamol showed bronchoprotection at 1 h but not at 3 h post-dosing whereas maximal concentrations of formoterol and salmeterol showed protection up to 5 h post-aqueous-aerosol dosing, giving a duration order of salmeterol > formoterol > salbutamol. All three α₂-adrenoceptor agonists showed dose-dependent bronchoprotection and duration of action following intravenous administration; salbutamol and salmeterol were equipotent and both were less potent than formoterol. Bronchoprotection obtained with sub-maximal concentrations of all three α₂-adrenoceptor agonists faded within 30 min following iv administration, but this could be extended by increasing the doses. These results demonstrate that the route of administration is important in determining the duration of action of α₂-adrenoceptor agonists in the lung. Furthermore, such findings lend support to the suggestion that the physico-chemical characteristics of salmeterol govern its duration of action rather than sustained binding of this agonist to a α₂-adrenoceptor exo-site.     

Role of the 5-HT(2A)receptor and alpha(1)-adrenoceptor in the contractile response of rat pulmonary artery to 5-HT in the presence and absence of nitric oxide

This study investigated the role of 5-HT(2A)receptors and alpha(1)-adrenoceptors in the contractile response to 5-HT in the first branch pulmonary artery of the rat and their interaction with endogenous nitric oxide. 5-HT and phenylephrine induced concentration-dependent contractions. The alpha(1)-adrenoceptor antagonists prazosin, HV723 and phentolamine produced concentration-dependent rightward shifts of the 5-HT concentration-response curves (CRC) consistent with an action at alpha(1)-adrenoceptors. The 5-HT(2)receptor antagonists ritanserin, ketanserin and methysergide produced rightward shifts that were less than would have been predicted for an action solely at 5-HT(2A)receptors. 5-HT and phenylephrine CRCs were shifted to the left by l -NAME. Endothelium denudation also increased the tissue sensitivity to 5-HT. In the presence of l -NAME, ketanserin produced greater antagonism of the 5-HT CRC but not the phenylephrine CRC. Ketanserin also produced greater antagonism of the 5-HT CRC in endothelium denuded rings compared with endothelium intact rings. These findings indicate (a) that both the alpha(1)-adrenoceptor class and the 5-HT(2A)receptor is involved in the contractile response to 5-HT; (b) in the presence of endogenous nitric oxide the contractile response to 5-HT is mediated predominently by alpha(1)-adrenoceptors; (c) inhibition of endogenous nitric oxide potentiates the 5-HT(2A)receptor-mediated component of the contraction.     

Autocrine Regulation of TGF β Expression in Adult Cardiomyocytes

G. Taimor, K.-D. Schlüter, K. Frischkopf, M. Flesch, S. Rosenkranz and H. M. Piper. Autocrine Regulation of TGF β Expression in Adult Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1999)31 , 2127–2136. As shown before, TGF β acts in an autocrine manner on the induction of hypertrophic responsiveness to β -adrenoceptor stimulation in cultured ventricular cardiomyocytes of adult rat. We now investigated how TGF β expression and activation is regulated in these cultures and how β -adrenoceptor stimulation influences TGF β -mRNA expression. It was found that freshly isolated cardiomyocytes secrete latent TGF β in the culture medium. Supplementation of the cultures with 20% FCS resulted in activation of the secreted TGFβ to 4.1±0.2 ng/ml active TGF β after 6 days. Presence of the protease inhibitor aprotinin (50μ g/ml) reduced TGF β activity by 44±5% (n=5, P<0.05). In cultures supplemented with 5% FCS, TGF β was not activated. Active TGF β downregulated its mRNA-expression: after 6 days TGF β₁-mRNA was reduced to 55.1±11.0%, TGFβ₂ -mRNA to 30.1±16.5%, and TGF β₃-mRNA to 0.3±0.4% in 20% FCS-cultures as compared to their expression in freshly isolated cells (n=4, P<0.05). TGFβ -mRNA expression did not change in cultures without active TGF β. Isoprenaline (1 μ m) increased TGF β₁-mRNA only in cultures which had been pre-exposed to active TGF β. This effect was also seen when hearts from normal mice were compared with hearts from transgenic mice overexpressing TGFβ₁ : only in hearts from transgenic animals perfusion with isoprenaline increased TGFβ₁ -mRNA. In conclusion, isolated cardiomyocytes release latent TGF β, which is activated by external proteases. Active TGF β downregulates its own mRNA expression. Preexposure to TGF β is necessary for a β -adrenoceptor-mediated increase in TGF β₁-mRNA in cardiomyocytes.     

Increased Expression of 5-HT3 and NK1 Receptors in 5-Fluorouracil-Induced Mucositis in Mouse Jejunum

Background and Objective

Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80 % of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT₃ and NK₁ receptor expression by immunohistochemical analysis.

Methods

5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay.

Results

The numbers of 5-HT₃ receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT₃ receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK₁ receptor cells in mucosa and immunopositive expression of NK₁ receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT_3A, 5-HT_3B, and NK₁ receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT₃ or NK₁ receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT₃ and NK₁ receptors colocalized with CD11b.

Conclusions

The 5-HT₃ and NK₁ immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

Functional 5-HT receptor subtypes in the isolated canine common carotid artery

Summary The vascular responses to 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT, a selective 5-HT₁-like receptor agonist), alphamethyl-5-HT (-M-5-HT, a relatively selective 5-HT₂ receptor agonist), noradrenaline (NA), and KCl were examined in isolated, cannulated, and perfused canine common carotid arterial preparations. They caused strong vasoconstrictions. The rank order of vasoconstrictive potency was 5-HT > -M-5-HT NA > 5-CT >> KCl. The 5-HT-induced vasoconstriction was significantly depressed by methysergide (a 5-HT₁ and 5-HT₂ receptor antagonist), ketanserin (a selective 5-HT₂ receptor antagonist), and spiperone (a selective 5-HT₂ receptor antagonist). The 5-CT- and -M-5-HT-induced vasoconstrictions were also significantly inhibited by methysergide, spiperone, and ketanserin. The NA-induced vasoconstriction was readily inhibited by bunazosin (an -adrenoceptor antagonist) and ketanserin but not significantly inhibited by spiperone and methysergide. KCl has a weak potency for producing a vasoconstriction of the canine common carotid artery. A relatively large dose of diltiazem (a calcium channel blocker) did not modify 5-HT-induced vasoconstrictions. From these results, we conclude that (a) the canine common carotid artery contains abundant 5-HT receptors; (b) there are no functional 5-HT₁ receptors, but 5-HT₂ receptors are prominent; (c) 5-CT-induced vasoconstrictions might be due to activation of 5-HT₂ but not to 5-HT₁ receptors; (d) 5-HT-induced vasoconstriction might not involve -adrenoceptors; and (e) the vasoconstriction related to 5-HT in the common carotid artery is not significantly mediated via activation of calcium ion channels of smooth muscle cells, but may be induced by calcium ions from intracellular stores.     

Effects of Maternal Ethanol Consumption and Buspirone Treatment on 5-HT1A and 5-HT2A Receptors in Offspring

In utero ethanol exposure results in a decreased concentration of serotonin (5-HT) in brain regions containing the cell bodies of 5-HT neurons and their cortical projections. The concentration of 5-HT reuptake sites is also reduced in several brain areas. The present study extended prior work by evaluating the effects of chronic maternal ethanol consumption and maternal buspirone treatment on 5-HT_1A and 5-HT_2A receptors in multiple brain areas of offspring. Receptors were quantitated early in postnatal development and at an age when the 5-HT networks are normally well-established. Because fetal 5-HT functions as an essential neurotrophic factor, these studies also determined whether treatment of pregnant rats with buspirone, a 5-HT_1A agonist, could overcome the effects of the fetal 5-HT deficit and prevent ethanol-associated receptor abnormalities. The results demonstrated that in utero ethanol exposure significantly alters the binding of 0.1 nM [³H]-8-hydroxy-dipropylaminotetralin to 5-HT_1A receptors in developing animals. Ethanol impaired the development of 5-HT_1A receptors in the frontal cortex, parietal cortex, and lateral septum; these receptors did not undergo the normal developmental increase between postnatal days 19 and 35. The dentate gyms was also sensitive to the effects of in utero ethanol exposure. 5-HT_1A receptors were increased in this region at 19 days. Maternal buspirone treatment prevented the ethanol-associated abnormalities in 5-HT_1A receptors in the dentate gyms, frontal cortex, and lateral septum. Neither maternal ethanol consumption nor buspirone treatment altered the binding of 2 nM [³H]ketanserin to 5-HT_2A receptors in the ventral dentate gyrus, dorsal raphe, parietal and frontal cortexes, striatum, substantia nigra, or nucleus accumbens.     

Activation of Connexin-43 Hemichannels Can Elevate [Ca]iand [Na]iin Rabbit Ventricular Myocytes During Metabolic Inhibition

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na⁺and Ca²⁺accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca²⁺]_iand [Na⁺]_iin myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4μ m fluo-3 AM for 30 min, or with 5 μ m sodium green AM for 60 min at 37°C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 m NaCN, 20 m 2-deoxy- -glucose and 0-glucose); or (3) MI+H (0.95 m , 4.7 m ) for 60 min. Propidium iodide (PI, 25 μ m) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca²⁺]_iand [Na⁺]_iwere then calculated by calibration. In some experiments, the effect of 10 μ m tetrodotoxin (TTX) and 20 μ m nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca²⁺]_iand [Na⁺]_iin myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca²⁺]_iand [Na⁺]_iin myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca²⁺]_iand [Na⁺]_ioverload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na⁺and Ca²⁺channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.     

Anatomy of F1-ATPase powered rotation

F₁-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F₁ structures 2HLD1 and 1H8E (Protein Data Bank).The purified F₁-ATPase is a molecular motor that can hydrolyze ATP to drive counterclockwise (CCW) rotation of the γ-subunit within the (αβ)₃-ring (Fig. 1A). In most living organisms, the F_o component of the F_oF₁ complex uses energy derived from a proton-motive force across a membrane to power F₁-dependent synthesis of ATP from ADP and P_i. Consumption of an ATP at each F₁ catalytic site, primarily composed of a β-subunit, correlates with a 120° rotational power stroke of the γ-subunit separated by a catalytic dwell with an 8-ms duration in Escherichia coli enzyme (1). A second “ATP-binding” dwell can occur after the γ-subunit has rotated ∼30° to 40° from the catalytic at limiting substrate concentrations (2, 3). Thus, three successive catalytic events that include power strokes and dwells are required to complete one revolution of the γ-subunit. Once bound to F₁, ATP is retained for 240° (3) such that the ADP and P_i generated are released two catalytic events later.Open in a separate windowFig. 1.Structural components of the F₁-ATPase molecular motor. (A) Top (from membrane) and side views of F₁ composed of the ring of α (orange) and β (purple) subunits surrounding the γ-subunit rotor (blue and green). (B) Open (β_E) and closed (β_D) conformations of the catalytic site composed of the catalytic domain (tan ribbon) and the lever domain (purple) relative to the γ-subunit coiled-coil (green) and foot (blue) domains. In the Gibbons et al. (32) F₁ structure (PDB ID code 1E79) used here, the γ-subunit foot domain is rotated 7° CCW from the structure determined by Menz et al. (4).The γ-subunit is composed of coiled-coil and globular “foot” domains where the former extends through the core of the (αβ)₃-ring (Fig. 1B). The β-subunits contain a catalytic domain and a C-terminal “lever” domain that is extended or open when the catalytic site is devoid of nucleotide and contracted (closed) when nucleotide is bound. In most F₁ crystal structures (4, 5), the coiled-coil faces the β-subunit with the open lever (β_E) whereas the foot domain extends over the lever domains of catalytic sites that usually contain bound ADP (β_D) and ATP (β_T). Although crystal structures provide excellent pictures of the subunit conformations at one rotational position, the rotational movement of the γ-subunit between these static structures and the mechanism in which ATP fuels this movement occurs remain major unresolved questions.Consensus is currently lacking regarding the relationship of nucleotide occupancy at the three catalytic sites to the catalytic dwell and ATP-binding dwell despite the intense scrutiny this question has received since Boyer and coworkers (6, 7) showed that F₁ operates via an alternating site mechanism. The catalytic dwell includes ATP hydrolysis and is believed to be terminated by the release of phosphate (3). Some single-molecule experiments support a mechanism whereby ATP binding and ADP release are concurrent during the ATP-binding dwell (3). As a result, only two catalytic sites are occupied the majority of the time such that three-site occupancy occurs transiently during the ATP-binding dwell. However, these results are inconsistent with the F₁ structure that contains transition state analogs and has three-site nucleotide occupancy (4). Nucleotide binding studies also strongly support a mechanism in which all three sites must be occupied (8, 9) and are consistent with other single-molecule studies that support alternative three-site mechanisms (10–12). At this time there is no consistent evidence that correlates any of the crystal structures to the prevailing rotational mechanism.The β-subunit lever domain is positioned to push against the γ-foot and the γ–coiled-coil as it opens and closes, respectively (Fig. 1B). The asymmetry of the γ-subunit at these interfaces resembles a camshaft that would be consistent with CCW directionality in response to lever movement. The energy for a 120° power stroke has been proposed to derive from the binding affinity of ATP that is used as ATP binding-induced closing of the β-lever (13) and is supported by experiments in which the lever was truncated (14).Based on single-molecule measurements, it was concluded that F₁ is nearly 100% efficient (15). A necessary outcome of this conclusion is that the 120° power strokes must occur at a constant angular velocity (13). Although a number of simulation studies have modeled rotation of the F₁-ATPase γ-subunit (16–19), only one of these (19) has provided a result showing that the angular velocity should vary during a power stroke. The claim of 100% efficiency (15) that serves as the energetic basis of this power stroke mechanism is unwarranted because the magnitude of the viscous flow coupling to the surface was unknown owing to technical limitations, and the authors erroneously used the value of ΔG° in lieu of ΔG in their calculation. The technical handicap was subsequently overcome by Junge and coworkers (20, 21), who relied on elastic probe curvature instead of rotation speed to calculate the average torque of the power stroke. Similar average torque values were subsequently obtained using rotation under limiting drag conditions, when the drag on the probe was measured directly (22).Based on 100% enzyme efficiency, it was difficult to explain how the energy from ATP binding was able to power 120° of rotation when the catalytic dwell interrupts rotation 80° after ATP binds. It was hypothesized that the remaining binding energy needed to power the final 40° of rotation until the next ATP binds is stored as elastic energy in the closed β-lever, which upon product release pushes on the γ-foot as it opens (13). However, to date, experimental evidence that tests these hypotheses is lacking owing to the inability to measure the rotary motion under conditions where the angular velocity is limited by the internal mechanism of the motor.Here we have resolved the angular velocity as a function of the rotational position using an assay that provides 10-μs time resolution. The results clearly show that the angular velocity is not constant during a power stroke, but undergoes a series of accelerations and decelerations as a function of rotational position. The slower angular velocity observed with the lower-affinity substrate ITP provides direct evidence that substrate binding affinity provides energy to power rotation. The correlation of the angular velocity profile of the final 85° of rotation presented here to the profile resulting from the simulations of Pu and Karplus (19) strongly supports a model in which ATP binding-dependent closure of the lever applies force to the γ-subunit. This also provides evidence that associates the F₁ structures of Kabaleeswaran et al. (23) and Menz et al. (4) with the protein conformations at 35° and 120° because they were used as the reference structures for the simulations of Pu and Karplus (19). The data also show that elevated ADP concentrations increase dwells at ∼35° and decrease the angular velocity between 85° and 120°. This indicates that ADP can remain bound subsequent to the ATP-binding dwell consistent with a three-site mechanism.     

Venoconstrictor responses to dihydroergocristine and dihydroergotamine: Evidence for the involvement of 5-HT1 like receptors

Summary Dihydroergocristine (DHEC) and dihydroergotamine (DHE) were investigated on canine saphenous veins in vivo and on canine saphenous veins and basilar arteries in vitro. Following local IV infusion in vivo, the venoconstrictor response to DHEC was about 30% weaker than that produced by DHE. When administered orally, however, both ergot alkaloids elicited similar venoconstrictor effects. In vitro maximal contractile responses to DHEC and DHE of basilar arteries were only 20–30% of those produced by 5-HT, whereas in saphenous veins both DHEC and DHE elicited similar maximal effects as those observed with 5-HT. In saphenous veins, methiothepin antagonized venoconstrictor responses to 5-HT, DHEC, and DHE within the same concentration range, being significantly less potent when tested against noradrenaline. The reverse was true for yohimbine, which was significantly more potent against noradrenaline than againificantly more potent against noradrenaline than against 5-HT, DHEC, and DHE. It is suggested that the venoconstrictor responses to both DHEC and DHE are mediated through 5-HT₁-like receptors.     

Phosphorylation of Phospholamban at Threonine-17 in the Absence and Presence of β -Adrenergic Stimulation in Neonatal Rat Cardiomyocytes

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca²⁺signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca²⁺/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca²⁺channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca²⁺stores by ryanodine or thapsigargin in the absence or presence of β -adrenergic stimulation. The isoproterenol (0.1 μ )-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 μ S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 μ ) reduced both the isoproterenol (0.1μ ) and S(-)-Bay K8644-(1 μ ) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 μ ) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5±6.3% and 92.5±11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1±0.3% and 8.6±2.1%, respectively. However, the effect of KN-93 was attenuated (47.8±3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between β -adrenoceptor stimulation and intracellular Ca²⁺at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca²⁺transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.     

F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F₁-ATPase (F₁) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α₃β₃, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F₁ structure and the rotation angle of the motor. Remarkably, a structure of F₁ in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F₁-ATPase.ATP synthase (F₁F_o-ATPase) catalyzes ATP synthesis from ADP and P_i in cells. The isolated F₁ portion is called F₁-ATPase, because it also catalyzes the reverse reaction, ATP hydrolysis (1–3). The α₃β₃γ-catalytic core complex of F₁-ATPase (denoted F₁) is a rotary molecular motor in which three αβ-dimers are arranged around the central γ-shaft (4). Unidirectional rotation of γ is driven by the free energy derived from sequential ATP hydrolysis at catalytic sites in the three αβ-dimers (5–7). Under an external torque, F₁ synthesizes ATP coupled to the rotation of γ in the opposite direction (8). This reversible operation of F₁ is achieved by the tripartite mechanochemical coupling between chemical reactions at the catalytic sites of αβ, conformational changes in the stator ring-α₃β₃, and orientation of γ.A combination of the rotation assay (5, 6) and single-molecule fluorescence imaging techniques (9) has led to a detailed picture of the coupling between chemical reactions in α₃β₃ and the rotary angles (10, 11). One ATP hydrolysis reaction in α₃β₃ drives discrete 80° + 40° substeps of γ in bacterial F₁ (7). The 80° substep is mainly driven by the binding energy of ATP (7, 9). The dwell before the 80° substep is, therefore, named the ATP waiting dwell. Release of the product, ADP, occurs before completion of the 80° substep (9, 10). The angle-dependent affinity of ADP suggests that the ADP release event also contributes part of the energy for the 80° substep (10). The dwell before the 40° substep is called the catalytic dwell; it consists of two rate-limiting events: ATP cleavage and release of the product, P_i (10, 12). The 40° substep is accompanied by a decrease of P_i affinity, with release that, in turn, generates torque (10). The coupling scheme between chemical reactions in α₃β₃ and the rotary angles has, therefore, been almost completely established (10).In the coupling of chemical reactions and α₃β₃-conformations, the key concept is thought to be the binding change mechanism, in which three catalytic sites in F₁ undergo sequential transitions between conformational states with different affinities for nucleotides corresponding to different rotary angles (1). The binding change mechanism is supported by the first crystal structure of F₁, in which two βs adopt the closed form with nucleotides and the other β adopts the open form without a nucleotide (4). The 120° step of γ observed in the rotation assay further supports this mechanism (6).However, we still face significant gaps in the structural ATPase cycle. Previous studies have suggested that F₁ should adopt at least two distinct conformational states for the ATP waiting dwell (ATP waiting form) and the catalytic dwell (catalytic form) based on rotation and tilting angles of γ (13). Furthermore, based on indirect evidence, it has been pointed out that the first crystal structure should represent the catalytic form or forms similar to the catalytic intermediate states (14–18). Although the crystal structures of the α₃β₃γ-complex differ from each other in terms of their nucleotide binding states and detailed configurations of the residues, their global structures are similar to the first crystal structure (19), which leaves the structure of the ATP waiting form unresolved. Closing this gap in the conformational cycle will deepen our understanding of the coupling between chemical reactions, α₃β₃-conformations, conformations, and rotary angles, not least by providing critical input into the theoretical modeling of F₁ (20–26).Here, we use the FRET technique to elucidate the conformational transitions of α₃β₃-conformations in F₁. FRET involves excited-state energy transfer from one fluorescent dye (donor) to another (acceptor) through dipole–dipole interactions (27). Single-pair FRET measurements combined with single-molecule techniques have been used to investigate the dynamics of intramolecular conformational changes or intermolecular interactions at the single-molecule level (28–34), including for F_oF₁-ATP synthase (35–37). We perform single-molecule FRET measurement to monitor distance changes between two fluorescently labeled βs and simultaneously monitor the rotational steps of γ. The FRET data allow us to distinguish the ATP waiting form from the catalytic form and thus, relate these dwelling states to the respective crystal structures. A systematic comparison of the crystal structures reveals the structural basis of the ATPase cycle. This study provides a structural basis for tripartite coupling among chemical reactions, conformations in the stator, and rotary angles in bacterial F₁.     

Serotonin receptor subtypes influence prolactin secretion in the turkey

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D₁ dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(−)-DOI hydrochloride (DOI), a selective 5-HT_2A eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT_2A receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D₁ DA receptors in the INF were blocked by the D₁ DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT_2A receptor subtype. The DOI-induced PRL increase did not occur when (±)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T_1A receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT_2A receptors are activated, they influence the release of DA to the INF. When 5-HT_1A receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT_2A receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D₂ DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT_1A receptors, could effectively inhibit PRL secretion.     

Trapping the ATP binding state leads to a detailed understanding of the F1-ATPase mechanism

The rotary motor enzyme F_oF₁-ATP synthase uses the proton-motive force across a membrane to synthesize ATP from ADP and P_i (H₂PO₄⁻) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 10⁵. This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F₁-ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40° from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why P_i is not released immediately after ATP hydrolysis, but only after a subsequent 120° rotation, in agreement with experiment. A concerted conformational change of the α₃β₃ crown is shown to induce the 40° rotation of the γ-subunit only when the β_E subunit is empty, whereas with P_i bound, β_E serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F₁-ATPase achieves the coupling between the small changes in the active site of β_DP and the 40° rotation of γ.The molecular motor F_oF₁-ATP synthase is composed of two domains: a transmembrane portion (F_o), the rotation of which is induced by a proton gradient, and a globular catalytic moiety (F₁) that synthesizes and hydrolyzes ATP. The primary function of the proton-motive force acting on F_oF₁-ATP synthase is to provide the torque required to rotate the γ-subunit in the direction for ATP synthesis (1, 2). The catalytic moiety, F₁-ATPase, has an α₃β₃ “crown” composed of three α- and three β-subunits arranged in alternation around the γ-subunit, which has a globular base and an extended coiled-coil portion (3) (Fig. 1A). F₁-ATPase by itself binds ATP and hydrolyzes it to induce rotation of the γ-subunit (in the opposite direction from that for synthesis) on the millisecond time scale under optimum conditions (4, 5). All of the α- and β-subunits bind nucleotides, but only the three β-subunits are catalytically active. The original crystal structure (3) of F₁-ATPase from bovine heart mitochondria (MF₁) led to the identification of three conformations of the β-subunit: β_E (empty), β_TP (ATP analog bound), and β_DP (ADP bound); Fig. 1A. In the known structures of F₁-ATPase, which apparently are near the “catalytic dwell” state, the state in which catalysis occurs (6, 7), the β_E subunit conformation is partly to fully open and is very different from those of the β_TP and β_DP subunits, which are closed and very similar to each other (SI Appendix, SI1).Open in a separate windowFig. 1.(A) F₁-ATPase. The three β-subunits and the γ-subunit are shown (α-subunits are not shown for clarity): β_E (yellow), β_DP (orange), β_TP (gold), and γ (purple). To define the β_DP subunit conformation we use the angle between helix B (βT163-A176) and helix C (βT190-G204). The two helices are highlighted: helix B (blue) and helix C (gray); the B^C angle is depicted as a red angle. The β_DPH6 helix, whose orientation was reported to undergo a 20° change during the 40° substep γ-rotation, is highlighted as red. During the forced rotation simulations with an external torque, the force acts on the C_α atom of MF₁:γM25 (shown as a red sphere). The direction of the force is determined as the cross-product of the radial vector of γM25:C_α and the rotational axis (green). (B) Proposed 360° rotation cycle of F₁-ATPase showing the subunit conformations, as well as the binding–release of ligands and the hydrolysis of ATP. Starting from the binding of an ATP* to the β_E subunit in the ATP waiting state (0°), rotation of the γ-stalk by 200° (80°, 40°, 80°) leads to the transition of β_E (γ = 0°) via β_TP (γ = 80°) to β_DP (γ = 200°), the catalytic dwell state where hydrolysis of ATP* takes place. The hydrolysis product P_i* in the β_DP subunit is not released at this catalytic dwell (200°). Instead, the other hydrolysis product ADP* is released first after a 40° rotation [β_DP (200°) → β_HO (240°)]. Then, β_HO is transformed to β_E and P_i* is released after an additional 80° rotation to another catalytic dwell state (320°); the latter is shown in brackets outside the main cycle (see below). Finally, the release of P_i* from β_E leads to a 40° rotation that completes the 360° cycle (21, 41). The other subunits are going through corresponding cycles offset by 120° (β_DP) and 240° (β_TP), respectively. Here, the prime symbol when it appears on the β_DP and β_E conformations indicates that the conformation of corresponding subunits change slightly in or near the specified reaction steps. The γ-subunit is shown as a yellow oval, and its rotation during the hydrolysis cycle is indicated by a red arrow. The reaction steps occurring in or near the catalytic dwell and corresponding changes of ligands in each β-subunit are also shown in the 320° catalytic dwell: The first state (Left in the 320° catalytic dwell) has a bound ATP in β_DP′, and is thus referred to as a prehydrolysis state (the state before the hydrolysis of ATP during the catalytic dwell). The second state (Middle) represents the state after ATP hydrolysis (posthydrolysis state), and the third state (Right) presents the state after the release of P_i bound in β_E′ (postrelease state).     

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB₁. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB₁—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB₁ and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB₁ structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.Classically, our understanding of G protein-coupled receptor (GPCR) signaling presumed that the receptor formed one unique, active receptor structure in response to agonist binding. We now know that this paradigm is too simple. A growing body of evidence shows that GPCRs can adopt different active conformations depending on the type of signal (ligand) bound, making it unlikely that only one GPCR structure is present at any given moment (1, 2). These different ligand-dependent conformations could explain why a wide range of activities can be observed for some GPCRs, such as coupling to multiple different G-protein types or signaling through non–G-protein signaling partners, such as the protein arrestin (3). This phenomenon—diverse ligands bound to the same receptor selectively eliciting different signaling pathways—is referred to as functional selectivity or biased signaling.What are these different receptor conformations, and why might they result in biased signaling? One possibility is that they involve different orientations of transmembrane helix 5 (TM5) and TM6 in the cytoplasmic face. An outward movement of TM6 is critical for G-protein activation, because it exposes a hydrophobic binding site and enables formation of the ternary complex of receptor, ligand, and G protein (4–9). Newer evidence suggests that there is likely some plasticity in TM6 movement during activation, with differences in either the magnitude or probability of the movement explaining varying degrees of G-protein signaling (3, 10, 11).Some types of biased signaling may also arise when TM7 and its attached helix 8 (H8) adopt different conformations in the cytoplasmic face, because movements in this region have been detected during receptor activation (12–14). However, H8/TM7 movement does not seem to be required for G-protein activation (15), and this region does not contact the G protein in the recent ternary complex structure (7). For these reasons, H8/TM7 movements may not be directly involved in G-protein binding but rather, may play a role in the binding of arrestin and/or kinase, thus triggering arrestin-centric signaling pathways (14, 16).The mechanism(s) through which allosteric molecules alter GPCR structure is also an unresolved question and an area of increasing interest (17–19) for which novel approaches are being developed (20) because of the potential that these ligands offer for new treatment paradigms (21). Allosteric ligands for several GPCRs have now been identified, including ligands for the cannabinoid, muscarinic, and μ-opioid.To gain more information about the structural changes accompanying both biased signaling and allosteric modulation of GPCRs, we have been studying the effects of an unusual allosteric ligand on CB₁, the marijuana GPCR. The use of this ligand, called Org 27569, provides a unique way to detect previously unidentified GPCR conformations for several reasons. First, because it binds allosterically, Org 27569 likely uses a different mechanism to act on CB₁. It also enables well-characterized radioactively labeled orthosteric CB₁ ligands to be used. Second, Org 27569 exhibits a number of unusual effects—it increases agonist binding to the receptor while simultaneously inhibiting G-protein activation (10, 22), and inducing biased signaling (23–25). Thus, it is hard to imagine how these different effects could occur unless the CB₁–Org 27569 bound state adopts a unique and different conformation. A cartoon representation of CB₁ and the putative Org 27569 binding site is shown in Fig. 1.Open in a separate windowFig. 1.A 2D cartoon model of CB₁ showing the approximate location of orthosteric and allosteric binding sites and the various mutations used. The traditional (orthosteric) ligand binding site is depicted as a dashed white oval, and the (proposed) allosteric ligand Org 27569 binding site is depicted as a dashed purple circle (50). Key point mutations in CB₁ include a CAM (I348Y^6.40; green) or CIM (Y294A^5.58; red). These mutations presumably cause their effect by altering interactions with a highly conserved Arg (R) in TM3 (black square). Radioligand binding studies used CB₁-Gα_i, a full-length human CB₁ receptor fused to the G-protein Gα_i (tan). SDFL studies used a minimal cysteine receptor (θ) with a truncated N terminus (Δ87) and a truncated C terminus (Δ417) to which the 1D4 epitope tag (black boxes) is attached to enable purification and unique reactive cysteines introduced at either TM6 (A342C^6.34; blue) or H8/TM7 (L404C^8.50; orange) to enable labeling with the fluorophore bimane (Inset). The different C-terminal modifications following residue 417 are indicated as X, where the sequence is either X₁ (for CB₁-Gα_i) or X₂ (for θ).Recently, we reported that, although Org 27569 stabilizes CB₁ interactions with the agonist, it simultaneously blocks the TM6 movements required for G-protein activation discussed above (10), thus explaining the negative effect of Org 27569 on G-protein signaling. These conclusions were based on site-directed fluorescence labeling (SDFL) studies of purified CB₁ that showed that, although Org 27569 induces CB₁ to adopt a high-affinity agonist binding conformation, it is not the high-affinity agonist binding conformation traditionally associated with the formation of the ternary complex with G protein (4).Here, we set out to characterize the conformation and energetics underlying this unique Org 27569 trapped state and identify a mechanism for the unusual effects discussed above using a combination of classical pharmacology, mathematical modeling, and SDFL studies. One goal was to determine if Org 27569 could act on the receptor in the absence of an orthosteric ligand. Another goal was to explore the linkage between Org 27569 binding and TM6 movements in CB₁ by asking the question: because Org 27569 binding blocks TM6 movement, does impairing TM6 movement inhibit Org 27569 binding? We did this by creating and testing two different CB₁ mutants: one constitutively active mutant (CAM) and one constitutively inactive mutant (CIM). In these mutants, TM6 movement was either enhanced (CAM) or impaired (CIM). Radioligand binding studies were then performed on these mutants in the presence of Org 27569 to test the above hypothesis and assess the energetics underlying Org 27569 binding. [All radioligand binding and efficacy measurements, unless otherwise stated, were done in the absence of sodium, a well-known negative allosteric modulator of GPCR activity, to enhance basal activity and reduce allosteric variables. This fact could contribute to the relatively high R*/R ratios that we observe for WT CB₁ along with the use of G-protein chimeras in our measurements (because G proteins can allosterically modulate receptor affinity).]Finally, we used SDFL to probe the structural differences between active, inactive, and Org 27569-bound CB₁, with the goal of identifying other structural changes in the receptor that might explain the mechanism of allosteric modulation and biased signaling, specifically focusing on movements at TM6 as well as H8/TM7. Our results are intriguing—they show that Org 27569 binding stabilizes a different receptor conformation, one that may be related to its ability to induce biased signaling.