D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins Dl, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone Hi or casein. Virtually all cyclin Dl-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin Dl-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin Dl-cdk4 complexes. Thus, cyclin Dl-associated kinase activity was not detected during the Go-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin Dl alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.D-and E-type cyclins govern the rate of progression of mammalian cells through the first gap phase (Gl) of the cell cycle and enforce the commitment of cells to replicate their chromosomal DNA (reviewed in reference 44). When quiescent cells enter the cell cycle, both classes of cyclins are synthesized during G, phase and associate with cyclin-dependent kinases (cdks) to form holoenzymes whose activities are presumed to facilitate entry into S phase.Cyclin E, first identified by complementation of G, cyclin deficiency in yeasts (22,25), is synthesized in late G, phase and associates with cdk2 to generate a histone Hi kinase that is maximally active during the Gl-to-S phase transition in mammalian cells (10, 23). The enforced overexpression of cyclin E in fibroblasts shortens their G, interval, decreases (but does not eliminate) their dependency on serum growth factors, and leads to a reduction in their size, indicating that cyclin E can be rate limiting for G1 progression (33). Its catalytic partner, cdk2, is also necessary for S-phase entry (34, 48), but it remains unclear whether this is due to its association with cyclin E and/or with cyclin A, which is expressed somewhat later during the cell cycle and steadily accumulates during the S and G2 phases (30,38,39). The activity of the cyclin E-c...

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We characterized an activation mechanism of the human LTRPC2 protein, a member of the transient receptor potential family of ion channels, and demonstrated that LTRPC2 mediates Ca2+ influx into immunocytes. Intracellular pyrimidine nucleotides, adenosine 5'-diphosphoribose (ADPR), and nicotinamide adenine dinucleotide (NAD), directly activated LTRPC2, which functioned as a Ca2+-permeable nonselective cation channel and enabled Ca2+ influx into cells. This activation was suppressed by intracellular adenosine triphosphate. These results reveal that ADPR and NAD act as intracellular messengers and may have an important role in Ca2+ influx by activating LTRPC2 in immunocytes.

Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca 2؉ levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT 3 receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.gastrointestinal tract ͉ RIN14B T he gastrointestinal tract has many functions, such as secretion, motility, and absorption. These functions are affected by various signals from the luminal contents, including nutrient and non-nutrient chemicals, mechanical factors, and microorganisms (1). The endocrine cells of the gut (hereafter enteroendocrine cells) are thought to be highly specialized mucosal cell subpopulations that receive luminal signals. There are more than 10 different types of enteroendocrine cells, and each type produces distinct transmitters/hormones (2). Serotonin (5-HT)-containing enterochromaffin (EC) cells, which are located throughout the gut, are considered to be the most prevalent enteroendocrine cells (3, 4). The 5-HT released from EC cells activate the submucosal sensory branch of the enteric nervous system and also control gastrointestinal motility and chloride secretion via interneurons and motor neurons (5, 6). Hence, EC cells are considered to be a major component of both the physiology and pathophysiology of gastrointestinal function (7,8). It has been suggested that EC cells respond to the contents of the lumen through the activation of receptor-operated or voltage-dependent Ca 2ϩ channels (9), however, the details of the cellular and molecular mechanisms have not yet been clarified.Many ion channels, like the transient receptor potential (TRP) channels expressed in sensory neurons, respond to natural compounds, especially spices and herbal medicines. For example, the vanilloid receptor (TRPV1) responds to the plant component capsaicin (the pungent ingredient in chili peppers), which produces the psychophysical sensation of heat or burning, whereas TRPM8 responds to menthol (found in peppermint), which produces ...

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Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1-and PKR2-gene-disrupted mice (Pkr1 ؊/؊ and Pkr2 ؊/؊ , respectively). Phenotypic analysis indicated that not Pkr1 ؊/؊ but Pkr2 ؊/؊ mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2 ؊/؊ mice. In addition, the Pkr2 ؊/؊ mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2 ؊/؊ mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2 ؊/؊ mice. The phenotype of the Pkr2 ؊/؊ mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.