Biomedical subjects

Diethelm W Richter

Publications and source records attributed to Diethelm W Richter.

18 recordsLinked to original sources

PubMed2007

Breathing dysfunctions associated with impaired control of postinspiratory activity in Mecp2-/y knockout mice.

Rett syndrome (RTT) is an inborn neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 gene (MECP2). Besides mental retardation, most patients suffer from potentially life-threatening breathing arrhythmia. To study its pathophysiology, we performed comparative analyses of the breathing phenotype of Mecp2-/y knockout (KO) and C57BL/6J wild-type mice using the perfused working heart-brainstem preparation (WHBP). We simultaneously recorded phrenic and efferent vagal nerve activities to analyse the motor pattern of respiration, discriminating between inspiration, postinspiration and late expiration. Our results revealed respiratory disturbances in KO preparations that were similar to those reported from in vivo measurements in KO mice and also to those seen in RTT patients. The main finding was a highly variable postinspiratory activity in KO mice that correlated closely with breathing arrhythmias leading to repetitive apnoeas even under undisturbed control conditions. Analysis of the pontine and peripheral sensory regulation of postinspiratory activity in KO preparations revealed: (i) prolonged apnoeas associated with enhanced postinspiratory activity after glutamate-induced activation of the pontine Kölliker-Fuse nucleus; and (ii) prolonged apnoeas and lack of reflex desensitization in response to repetitive vagal stimulations. We conclude that impaired network and sensory mediated synaptic control of postinspiration induces severe breathing dysfunctions in Mecp2-/y KO preparations. As postinspiration is particularly important for the control of laryngeal adductors, the finding might explain the upper airway-related clinical problems of patients with RTT such as apnoeas, loss of speech and weak coordination of breathing and swallowing.

Animals↗

PubMed2006

Calcium-regulated potassium currents secure respiratory rhythm generation after loss of glycinergic inhibition.

Mutant oscillator mice (Glra1(spd -/-)) are characterized by a developmental loss of glycinergic inhibition. These mice die during the third postnatal week presumably due to gradually increasing disturbances of breathing and motor behaviour. Some irregular rhythmic respiratory activity, however, is persevered until they die. Here we analysed cellular mechanisms that compensate for the loss of glycinergic inhibition and contribute to the maintenance of the respiratory rhythm. In a medullary slice preparation including the pre-Bötzinger complex we performed a comparative analysis of after-hyperpolarizations following action potentials (AP-AHP) and burst discharges (burst-AHP) in identified respiratory neurons from oscillator and control mice. Both AHP forms were increased in neurons from oscillator mice. These changes were combined with an augmented adaptation of firing frequency. Assuming that oscillator mice might upregulate calcium-activated K currents (BKCa) in compensation for the loss of glycinergic inhibition, we blocked the big KCa conductances with iberiotoxin and verified that the respiratory rhythm was indeed arrested by BK channel blockade.

Action Potentials↗

PubMed2005

Lack of the Kir4.1 channel subunit abolishes K+ buffering properties of astrocytes in the ventral respiratory group: impact on extracellular K+ regulation.

Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1-/- mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1-/- mice, containing the pre-Bötzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.

Action Potentials↗

PubMed2005

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

Animals↗

PubMed2005

Palmitoylation of the 5-hydroxytryptamine4a receptor regulates receptor phosphorylation, desensitization, and beta-arrestin-mediated endocytosis.

The mouse 5-hydroxytryptamine4a (5-HT4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534-2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. Overexpression of GRK2 significantly reduced the receptor-promoted cAMP formation. The Cys328/329-Ser mutant, which is constitutively active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effectively desensitized and internalized via a beta-arrestin-2 mediated pathway compared with the wild-type 5-HT4a. In contrast, G protein activation, phosphorylation, desensitization, and internalization of the other palmitoylation-deficient receptor mutants were affected differently. These findings suggest that palmitoylation plays an important role in modulating 5-HT4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.

Animals↗

PubMed2005

Mitochondrial organization and motility probed by two-photon microscopy in cultured mouse brainstem neurons.

Two-photon microscopy of rhodamine 123-labeled mitochondria revealed that mitochondria of neurons cultured from mouse respiratory center form functionally coupled, dynamically organized aggregates such as chains and clusters, while single mitochondria were rarely seen. Mitochondrial chain structures predominate in dendrites, while irregularly shaped mitochondrial clusters are mostly found in the soma. Both types of mitochondrial structures showed chaotic Brownian motions and the mitochondrial chains also revealed well-directed movements. The latter dislocations were arrested upon mitochondrial depolarization or blockade of mitochondrial ATP synthesis. Depolymerization of microtubules by colchicine or nocodazole or inhibition of protein phosphatases by calyculin A disrupted mitochondrial chains and the mitochondria accumulated in the soma. Forskolin and IBMX reversibly blocked directed movements of mitochondria, but did not affect their overall spatial distribution. Thus, protein phosphorylation seems to control both mitochondrial transport and organization. Protein phosphorylation downstream of enhanced cytosolic cAMP levels apparently regulates the transition from motile to non-motile mitochondria, while phosphorylation resulting from inhibition of types 1 and 2A protein phosphatases massively disturbs mitochondrial organization. The complex phosphorylation processes seem to control the close interaction of mitochondria and cytoskeleton which may guarantee that mitochondria are immobilized at energetic hot spots and rearranged in response to changes in local energy demands.

Animals↗

PubMed2004

Diversity of functional astroglial properties in the respiratory network.

A population of neurons in the caudal medulla generates the rhythmic activity underlying breathing movements. Although this neuronal network has attracted great attention for studying neuronal aspects of synaptic transmission, functions of glial cells supporting this neuronal activity remain unclear. To investigate the role of astrocytes in the respiratory network, we applied electrophysiological and immunohistochemical techniques to characterize astrocytes in regions involved in the generation and transmission of rhythmic activity. In the ventral respiratory group and the hypoglossal nucleus (XII) of acutely isolated brainstem slices, we analyzed fluorescently labeled astrocytes obtained from TgN(GFAP-EGFP) transgenic mice with the whole-cell voltage-clamp technique. Three subpopulations of astrocytes could be discerned by their distinct membrane current profiles. A first group of astrocytes was characterized by nonrectifying, symmetrical and voltage-independent potassium currents and a robust glutamate transporter response to d-aspartate. A second group of astrocytes showed additional A-type potassium currents, whereas a third group, identified by immunolabeling for the glial progenitor marker NG2, expressed outwardly rectifying potassium currents, smaller potassium inward currents, and only minimal D-aspartate-induced transporter currents. Astrocytes of all groups showed kainate-induced inward currents. We conclude that most of the astrocytes serve as a buffer system of excess extracellular glutamate and potassium; however, a distinct cell population (NG2-positive, A-type potassium currents) may play an important role for network plasticity.

Amino Acid Transport System X-AG↗

PubMed2003

Commentary on eupneic breathing patterns and gasping.

The term "eupneic activity pattern" is a trivial phenotypical description of a particular activity pattern in respiratory nerves as recorded under in vivo like experimental conditions. This term is, however, inadequate, because Eupnea describes a behavioral breathing performance that is trouble-free occurring without conscious effort. Obviously, the term "eupneic activity pattern" is meant to describe a neural activity that is normal and comparable with quiet breathing conditions. The various in vivo, in situ and in vitro preparations all generate their specific "normal" activity patterns, when the conditions are undisturbed. The commentary describes some of the numerous reasons why such normal activity patterns must be different in the various preparations without indicating their pathological operation. The conclusion is that special considerations are necessary for any extension of the in vitro and in situ findings into in vivo situations, because the capacity of the respiratory network is greatly reduced and thus not comparable with conditions leading to "eupneic breathing" in the fully intact animal.

Animals↗

PubMed2003

Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality.

The glycine transporter subtype 2 (GlyT2) is localized in the axon terminals of glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.

Amino Acid Transport Systems, Neutral↗

PubMed2003

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein.

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.

Amino Acid Sequence↗

PubMed2003

5-HT4(a) receptors avert opioid-induced breathing depression without loss of analgesia.

Opiates are widely used analgesics in anesthesiology, but they have serious adverse effects such as depression of breathing. This is caused by direct inhibition of rhythm-generating respiratory neurons in the Pre-Boetzinger complex (PBC) of the brainstem. We report that serotonin 4(a) [5-HT4(a)] receptors are strongly expressed in respiratory PBC neurons and that their selective activation protects spontaneous respiratory activity. Treatment of rats with a 5-HT4 receptor-specific agonist overcame fentanyl-induced respiratory depression and reestablished stable respiratory rhythm without loss of fentanyl's analgesic effect. These findings imply the prospect of a fine-tuned recovery from opioid-induced respiratory depression, through adjustment of intracellular adenosine 3',5'-monophosphate levels through the convergent signaling pathways in neurons.

Analgesics, Opioid↗

PubMed2003

Blockade of astrocyte metabolism causes delayed excitation as revealed by voltage-sensitive dyes in mouse brainstem slices.

Fluoroacetate is known to block cell metabolism and to change potassium conductances selectively in astrocytes. In a functional neuronal network with ongoing activity, we investigated the effects of such a blockade of the astrocytic metabolism by fluoroacetate on neuronal signal propagation. Transverse 400- microm slices were prepared from the caudal medulla of mice of postnatal day 3-8, which contained the hypoglossal nucleus receiving excitatory synaptic input from the ventral respiratory group. Propagation of excitation within this network was measured by optical imaging using the voltage-sensitive dye RH 795. A 464-element photodiode array allowed fast recordings of voltage changes within a small population of cells. The spatial and temporal resolution was advanced to 32 microm and 1.27 ms, respectively. Changes of cellular membrane potential levels were expressed as relative changes of fluorescence (DeltaI/I). Stimulus-evoked excitation of neurons propagating from the ventral respiratory group to the hypoglossal nucleus peaked after 7.2+/-0.6 ms ( n=6). The latency of this early excitatory response is consistent with the time course of stimulus-evoked EPSPs in whole-cell recordings. Mean changes of fluorescence in the hypoglossal nucleus were -2.1+/-0.5 x 10(-3) (DeltaI/I). After incubation in 1 mM fluoroacetate, the early depolarization was reduced to 69.1+/-9.8% of control ( n=6, p=0.034). Additionally, fluoroacetate induced a delayed excitatory response, such that fluorescence intensity did not return to baseline within 1s. Propagation velocity and spatial distribution of the voltage signal were not affected by fluoroacetate. Our results suggest that blockade of astrocyte metabolism impairs fast synaptic transmission and induces a delayed excitation, probably resulting from the combination of reduced repolarization of neurons and persistent depolarization of astrocytes.

Animals↗

PubMed2003

Serotonin receptors: guardians of stable breathing.

Disturbances of breathing arising from failures of the respiratory center are not uncommon. Among them, breath holding and apnea occur most frequently as consequences of pulmonary and cardiac diseases, hypoxia, head trauma, cerebral inflammatory processes, genetic defects, degenerative brain diseases, alcoholism, deep anesthesia and drug overdose. They are often life-threatening and fail to respond to existing pharmacotherapies. After extensive research, there is now a reliable basis for new strategies to treat respiratory disturbances by pharmacological manipulation of intracellular signaling pathways, particularly those involving the serotonin receptor family. Specific activation of these pathways effectively prevails respiratory disturbances and can be extended to treatment of life-threatening respiratory disorders in patients.

Animals↗

PubMed2002

5-Hydroxytryptamine 4(a) receptor is coupled to the Galpha subunit of heterotrimeric G13 protein.

Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter that regulates multiple events in the central nervous system. Many of the 5-HT functions are mediated via G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins, including G(s), G(i), and G(q) subfamilies (Martin, G. R., Eglen, R. M., Hamblin, M. W., Hoyer, D., and Yocca, F. (1998) Trends Pharmacol. Sci. 19, 2-4). Here we show for the first time that the 5-hydroxytryptamine 4(a) receptor (5-HT(4(a))) is coupled not only to heterotrimeric G(s) but also to G(13) protein, as assessed both by biochemical and functional assays. Using reconstitution of 5-HT(4(a)) receptor with different G proteins in Spodoptera frugiperda (Sf.9) cells, we have proved that agonist stimulation of receptor-induced guanosine 5'-(3-O-thio)triphosphate binding to Galpha(13) protein. We then determined that expression of 5-HT(4(a)) receptor in mammalian cells induced constitutive- as well as agonist-promoted activation of a transcription factor, serum response element, through the activation of Galpha(13) and RhoA. Finally, we have determined that expression of 5-HT(4(a)) receptor in neuroblastoma x glioma NIE-115 cells cause RhoA-dependent neurite retraction and cell rounding under basal conditions and after agonist stimulation. These data suggest that by activating 5-HT(4(a)) receptor-G(13) pathway, serotonin plays a prominent role in regulating neuronal architecture in addition to its classical role in neurotransmission.

3T3 Cells↗

PubMed2002

Effects of functional knock-out of alpha 1 glycine-receptors on breathing movements in oscillator mice.

The effects of a deficiency of glycinergic inhibition deriving from mutations of the glycine-receptor gene Glra1 on the breathing pattern of oscillator mice were studied. We compared the development of breathing frequency, tidal volume and minute ventilation from control mice (wild type- and heterozygous oscillator mice) with those of homozygous oscillator mice during early postnatal periods from p9 until p21. The changes of ventilation were correlated with body-weight and changes in blood-pH. During the second to third weeks of postnatal development, breathing frequency increased from 310 to 445.4 mm-1 in control mice. Oscillator mice reached a maximal value of 313.3 min-1 at p18 followed by a fast decrease to 233.0 min-1. This decrease is caused by a prolongation of expiratory duration. Tidal volume showed a steady increase from 6.6 to 15.1 microliters in control animals. In comparison, oscillator mice showed significant lower values after p14. After p15, minute ventilation of oscillator mice declined as compared with control animals leading to respiratory acidosis at p20.

Animals↗

PubMed2002

Differential modulation of I(h) by 5-HT receptors in mouse CA1 hippocampal neurons.

CA1 pyramidal neurons of the hippocampus express various types of serotonin (5-HT) receptors, such as 5-HT(1A), 5-HT(4) and 5-HT(7) receptors, which couple to Galpha(i) or Galpha(s) proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization-activated current I(h). Activation of 5-HT(1A) receptors induced an augmentation of current-induced hyperpolarization responses, while the responses declined after 5-HT(4) receptors were activated. The resting potential of neurons hyperpolarized (-2.3 +/- 0.7 mV) after 5-HT(1A) receptor activation, activation of 5-HT(4) receptors depolarized neurons (+3.3 +/- 1.4 mV). Direct activation of adenylyl cyclase (AC) by forskolin also produced a depolarization. In voltage clamp, the Ih current was identified by its characteristic voltage- and time-dependency and by blockade with CsCl or ZD7288. Activation of 5-HT(1A) receptors reduced I(h) and shifted the activation curve to a more negative voltage by -5 mV at half-maximal activation. Activation of 5-HT(4) and 5-HT(7) receptors increased I(h) and shifted the activation curve to the right by +5 mV. Specific activation of 5-HT(4) receptors by BIMU8 increased membrane conductance and showed an increase in I(h) in a subset of cells, but did not induce a significant alteration in the activation curve. In order to verify spatial differences, we applied BIMU8 selectively to the soma and to the dendrites. Only somatic application induced receptor activation. These data are confirmed by immunofluorescence stainings with an antibody against the 5-HT(4) receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5-HT(7) receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons.

Animals↗

PubMed2001

The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.

We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.

Acylation↗

PubMed2001

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel.

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 microM and 5 microM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.

Adenylyl Cyclases↗