Pilot study of the efficacy of renzapride on gastrointestinal motility and symptoms in patients with constipation-predominant irritable bowel syndrome.

AIM: To investigate the efficacy and safety of renzapride, a potent 5-hydroxytryptamine type-4 receptor full agonist and 5-hydroxytryptamine type-3 receptor antagonist in patients with constipation-predominant irritable bowel syndrome. METHODS: In this dose-escalating pilot study, 17 patients with constipation-predominant irritable bowel syndrome received placebo, renzapride 2 mg o.d. and renzapride 2 mg b.d. sequentially for 28 days. Response was determined by radio-opaque marker measurement of overall gastrointestinal and segmental colonic transit and patients' assessment of their irritable bowel syndrome symptoms. RESULTS: Renzapride reduced mean overall gastrointestinal transit time (placebo, 2.9 +/- 1.6 days; renzapride 2 mg o.d., 2.6 +/- 1.4 days; renzapride 2 mg b.d., 1.9 +/- 1.6 days) (P = 0.024) and accelerated segmental colonic transit, with statistically significant differences for renzapride 2 mg b.d. over placebo in caecum/ascending colon (P = 0.019) and descending colon (P = 0.022). Renzapride also reduced abdominal pain, increased the number of pain-free days and improved stool consistency. The frequency of reported adverse events was similar on renzapride and placebo. CONCLUSIONS: Renzapride is well-tolerated, stimulates gastrointestinal transit and improves symptoms in patients with constipation-predominant irritable bowel syndrome, particularly at the 2 mg b.d. dose, where improvements in gastrointestinal symptoms were evident over placebo. This study has established proof of concept and supports further investigation of renzapride in patients with constipation-predominant irritable bowel syndrome.

A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2.

The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The beta(2) adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Ca(v)1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate-dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein-coupled receptor.

Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase.

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.

Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor.

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca2+ influx through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIalpha by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca2+ influx but also close to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP.

SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit.

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.

Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest.

Human cyclin G2 together with its closest homolog cyclin G1 defines a novel family of cyclins (Horne, M. C., Goolsby, G. L., Donaldson, K. L., Tran, D., Neubauer, M., and Wahl, A. F. (1996) J. Biol. Chem. 271, 6050-6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor beta1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5-14-fold over control cell levels. Unstimulated immature B cell lines (WEHI-231 and CH31) and unstimulated or IgM B cell receptor (BCR) -stimulated mature B cell lines (BAL-17 and CH12) rapidly proliferate and express low levels of cyclin G2 mRNA. In contrast, BCR-stimulated immature B cell lines undergo growth arrest and coincidentally exhibit an approximately 10-fold increase in cyclin G2 transcripts and a decrease in cyclin D2 message. Costimulation of WEHI-231 and CH31 cells with calcium ionophores and protein kinase C agonists partially mimics anti-IgM stimulation and elicits a strong up-regulation of cyclin G2 mRNA and down-regulation of cyclin D2 mRNA. Signaling mutants of WEHI-231 that are deficient in the phosphoinositide signaling pathway and consequently resistant to the BCR stimulus-induced growth arrest did not display a significant increase in cyclin G2 or decrease in cyclin D2 mRNAs when challenged with anti-IgM antibodies. The two polyclonal activators lipopolysaccharide and soluble gp39, which inhibit the growth arrest response of immature B cells, suppressed cyclin G2 mRNA expression induced by BCR stimulation. These results suggest that in murine B cells responding to growth inhibitory stimuli cyclin G2 may be a key negative regulator of cell cycle progression.

Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue-specific and cell cycle-regulated expression.

We describe the isolation and characterization of cDNAs encoding full-length human and murine cyclin G1 and a novel human homologue of this cyclin designated cyclin G2. Cyclin G1 is expressed at high levels in skeletal muscle, ovary, and kidney. Following an initial up-regulation from early G1 to G1/S phase, cyclin G1 mRNA is constitutively expressed throughout the cell cycle in T and B cell lines. In contrast, in stimulated peripheral T cells, cyclin G1 mRNA is maximal in early G1 phase and declines in cell cycle progression. Cyclin G1 levels parallel p53 expression in murine B lymphocytes; however, in several human Burkitt's lymphomas, murine lymphocytes treated with transforming growth factor-beta, early murine embryos, and several tissues of p53 null mice, cyclin G1 levels are either inverse of p53 levels or expressed independent of p53. The cyclin G1 homologue, cyclin G2, exhibits 60% nucleotide sequence identity and 53% amino acid sequence identity with cyclin G1, and like cyclin G1, exhibits closest sequence identity to the cyclin A family. Distinct from cyclin G1, the amino acid sequence for cyclin G2 shows a PEST-rich sequence and a potential Shc PTB binding site. Cyclin G2 mRNA is differentially expressed compared to cyclin G1, the highest transcript levels seen in cerebellum, thymus, spleen, prostate, and kidney. In contrast to the constitutive expression of cyclin G1 in lymphocytes, cyclin G2 mRNA appears to oscillate through the cell cycle with peak expression in late S phase.

Assembly of the truncated immunoglobulin heavy chain D mu into antigen receptor-like complexes in pre-B cells but not in B cells.

Rearrangements of the IgH locus with JH joined to reading frame 2 of DH are greatly underrepresented in B cells. These rearrangements encode the truncated heavy chain D mu. In pre-B cells, we found D mu protein expressed on the cell surface and assembled into a complex with surrogate light chains, Ig alpha, and Ig beta. Cross-linking of either mu m- or D mu m- containing pre-B cell receptors triggered signal transduction reactions. In contrast, when expressed in mature B cell lines, D mu was not detected on the cell surface and did not efficiently bind kappa immunoglobulin light chains, but did associate with Ig alpha and Ig beta. These results characterize the interactions of D mu chain with other components of the B cell antigen receptor complex and suggest possible mechanisms by which D mu expression may interfere with B cell development.