Gap junction-mediated contraction of myoepithelial cells induces the peristaltic transport of sweat in human eccrine glands.

Eccrine sweat glands play an essential role in regulating body temperature. Sweat is produced in the coiled secretory portion of the gland, which is surrounded by obliquely aligned myoepithelial cells; the sweat is then peristaltically transported to the skin surface. Myoepithelial cells are contractile and have been implicated in sweat transport, but how myoepithelial cells contract and transport sweat remains unexplored. Here, we perform ex vivo live imaging of an isolated human eccrine gland and demonstrate that cholinergic stimulation induces dynamic contractile motion of the coiled secretory duct that is driven by gap junction-mediated contraction of myoepithelial cells. The contraction of the secretory duct occurs segmentally, and it is most prominent in the region surrounded by nerve fibers, followed by distension-contraction sequences of the excretory duct. Overall, our ex vivo live imaging approach provides evidence of the contractile function of myoepithelial cells in peristaltic sweat secretion from human eccrine glands.

Olfactory marker protein contains a leucine-rich domain in the Ω-loop important for nuclear export.

Olfactory marker protein (OMP) is a cytosolic protein expressed in mature olfactory receptor neurons (ORNs). OMP modulates cAMP signalling and regulates olfactory sensation and axonal targeting. OMP is a small soluble protein, and passive diffusion between nucleus and cytoplasm is expected. However, OMP is mostly situated in the cytosol and is only sparsely detected in the nuclei of a subset of ORNs, hypothalamic neurons and heterologously OMP-expressing cultured cells. OMP can enter the nucleus in association with transcription factors. However, how OMP is retained in the cytosol at rest is unclear. Because OMP is proposed to affect cell differentiation, it is important to understand how OMP is distributed between cytoplasm and nucleus. To elucidate the structural profile of OMP, we applied several bioinformatics methods to a multiple sequence alignment (MSA) of OMP protein sequences and ranked the evolutionarily conserved residues. In addition to the previously reported cAMP-binding domain, we identified a leucine-rich domain in the Ω-loop of OMP. We introduced mutations into the leucine-rich region and heterologously expressed the mutant OMP in HEK293T cells. Mutations into alanine increased the nuclear distribution of OMP quantified by immunocytochemistry and western blotting. Therefore, we concluded that OMP contains a leucine-rich domain important for nuclear transport.

Olfactory marker protein is unlikely to be cleaved by calpain 5.

Olfactory maturation marker protein (OMP) is expressed in olfactory receptor neurons and hypothalamic neurons. OMP is a nested gene located in the intron of calpain 5 (CAPN5), a Ca2+-dependent cysteine protease. Despite being located at the same genomic locus, genetic regulation of the reciprocal expression of OMP and CAPN5 has been suggested. By performing a motif search, we detected possible calpain cleavage sites in OMP. However, the direct proteolytic regulation of OMP by CAPN5 is unclear. Here, we generated OMP fused with Myc-tag and His-tag at its N- and C-termini and examined whether CAPN5 cleaves OMP into fragments by detecting immunoreactivity against Myc, OMP and His. Western blotting demonstrated that OMP was unlikely to be cleaved even in the presence of Ca2+ in vitro. We expressed OMP and CAPN5 in HEK293T cells and applied a calcium ionophore under physiological conditions in cellulo, which resulted in no apparent fragmentation of OMP. We also applied liquid chromatography/mass spectrometry to the electrophoresed fractions smaller than the uncut Myc-OMP-His signals, which demonstrated no significant fragmentation of OMP. These results collectively indicate that OMP is unlikely to be cleaved by CAPN5.

Olfactory receptor 78 is expressed in hypothalamic vasopressin/oxytocin neurons, parenchymal microglia and choroidal macrophages in mice.

Olfactory receptors have been detected in extraolfactory organs. Olfactory receptor 78 (Olfr78), proposed to respond to small organic acids, is widely expressed in the kidney, arterioles, colon, and prostate. However, its expression patterns in the brain remain largely unknown. Using immunohistochemistry, we revealed that Olfr78 was densely expressed in the hypothalamus and choroid plexus and sparsely expressed throughout the parenchyma. By costaining with cellular markers, we further found that Olfr78 was expressed in the somata and axons of vasopressin/oxytocin neurons in the hypothalamic paraventricular/supraoptic nuclei. Olfr78 was also strongly expressed in macrophages in the choroid plexus and moderately expressed in microglia near the parenchymal vasculature. Considering that these brain regions should communicate with cerebral blood flow, Olfr78 could contribute to sensing the humoral conditions surrounding the cerebrovascular system.

Olfactory marker protein interacts with adenosine nucleotide derivatives.

Olfactory marker protein (OMP) is a genetic signature for mature olfactory receptor neurons (ORNs). Recently, it has been proposed that OMP directly captures odour-induced cAMP to swiftly terminate the olfactory signal transduction to maintain neuronal sensitivity. In the present study, we show that OMP can also interact with other adenosine nucleotides as ATP, ADP and AMP with different affinities. We performed bioluminescent resonant energy transfer (BRET) assay to measure the binding actions of the adenosine nucleotide derivatives in competition to cAMP. Amongst all, ATP showed the bell-shape affinity to OMP in the presence of cAMP; ADP and AMP showed fewer affinities to OMP than ATP. In the absence of cAMP analogues, ATP alone bound to OMP in a dose dependent manner with a lower affinity than to cAMP. Thus, OMP possessed different affinities to ATP in the presence or absence of cAMP. OMP may interact differentially with ATP and cAMP depending on its supply and demand along the cAMP-associated signalling in the limited spaces of cilia of ORNs.

Olfactory marker protein elevates basal cAMP concentration.

Olfactory marker protein (OMP), which is expressed abundantly in mature olfactory receptor neurons, operates as a cAMP-binding protein. OMP captures phasic cAMP surges induced by sensory stimuli and punctuates the downstream signalling in the cilia. On the other hand, OMP is also abundant in the soma. At equilibrium, OMP should exhibit association/dissociation reactions with cAMP. To examine the steady-state function of OMP, we expressed OMP in an HEK293 heterologous expression system and measured the activity of cAMP-dependent protein kinase (PKA) using a cAMP response element/luciferase reporter assay. In the presence of OMP, the basal activity level of PKA was elevated to approximately twice as much as that in the absence of OMP. Upon tonic stimulation by membrane-permeable cAMP, the PKA activity increased in a dose-dependent manner and was greater in the presence of OMP at all doses until saturation. These results indicate that OMP, a cytosolic cAMP-binding protein, operates as a cAMP reservoir by increases the basal cAMP concentration and enhances tonic cAMP actions. Together with the previous finding that OMP acutely sequesters cAMP-related responses, these results indicate that OMP can buffer acute surges in cAMP and tonic production, which stabilizes the basal cAMP pool in the long run.

Olfactory marker protein captures cAMP produced via Gαs-protein-coupled receptor activation.

Olfactory marker protein (OMP) labels the matured stage of olfactory receptor neurons (ORN) and has promoted the investigation on the physiology of olfaction. OMP regulates olfactory sensitivity and axonal projection of ORNs, both of which are under the control of the olfactory signaling mediator cAMP. Recently, it has been reported that OMP contains cAMP-binding sites. OMP directly captures the photo-uncaged cAMP in the cytosol and rapidly terminates the olfactory cyclic nucleotide-gated (CNG) channels activity to sharpen the olfactory responses. Here, we investigate the contribution of OMP to cAMP acutely produced via activation of Gαs-protein coupled receptors (GPCR). We expressed OMP and non-desensitizing CNGA2 channels in HEK293T cells together with ß1-adrenergic receptors (ADRB1) or photo-sensitive ß2-adrenergic receptors (opto-ß2). Continuous puff of adrenergic agonist isoproterenol to HEK29T cells with ADRB1 induced the lasting CNGA2 currents in the absence of OMP, while OMP rapidly deactivated the CNGA2 channel activity with residual currents. Photo-activation of opto-ß2 in the absence of OMP induced the CNGA2 currents with a prolonged increase, while OMP swiftly deactivated the CNGA2 channels after the initial surge. Therefore, cytosolic OMP rapidly uncouples CNGA2 channels and cAMP-signaling produced via GPCRs in the submembrane compartment.

Olfactory marker protein directly buffers cAMP to avoid depolarization-induced silencing of olfactory receptor neurons.

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.

Olfactory receptor neurons express olfactory marker protein but not calpain 5 from the same genomic locus.

Gene expression is highly regulated to functionally diversify cells. Genes that cooperate in the same physiological processes occasionally reside within nearby regions in a chromosome. Olfactory marker protein (OMP) is highly expressed in mature olfactory receptor neurons (ORNs), but its physiological roles are not fully understood. According to the genomic map, the OMP gene is located within an intron of the calcium-dependent protease, calpain 5 (CAPN5); in other words, the OMP gene is a nested intronic gene. Thus, we attempted to investigate the gene expression and protein distribution of CAPN5 in the olfactory epithelium compared with that in the central nervous system (CNS). By performing reverse-transcriptase PCR and in situ hybridization, we confirmed that CAPN5 mRNA was expressed in the olfactory epithelium. We then performed immunohistological investigations using sliced preparations obtained from mice expressing GFP under OMP promoter activity. The detected GFP fluorescence was restricted to the knob, soma and axon bundles of the ORNs, while CAPN5 immunoreactivity (CAPN5-IR) was ubiquitously detected in the olfactory epithelial layer and lamina propria; signals were strongly detected in the supporting cells within the epithelium. In the CNS, CAPN5 signals were widely detected and were especially strong in the hippocampal formation and the piriform cortex as previously indicated. Therefore, these data indicate that ORNs express OMP but not CAPN5 from CAPN5 gene expression even though they are localized in the same genomic locus. The mechanisms by which the OMP promoter is regulated require detailed investigations.

Dual expression of constitutively active Gαs-protein-coupled receptors differentially establishes the resting activity of the cAMP-gated HCN2 channel in a single compartment.

The hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channel is a major subtype of the HCN channel family expressed in the nervous system that sets the membrane potential, regulates cell excitability and senses changes in the extracellular environment. Neurons express various Gαs-protein-coupled receptors (GPCRs), many of which show ligand-independent constitutive activity. These membrane-bound proteins are expressed in various subcellular compartments of neurons. Therefore, some proportion of HCN2 channels opens in response to the basal cAMP pool size produced by constitutively active GPCRs. Here, we employed an exogenous HEK293 expression system and voltage-clamp patch-clamp recordings to investigate basal HCN2 channel activity in the presence of two GPCRs with diverse basal activities in a single compartment. We utilized the ß2-adrenoceptor (ß2AR) together with odorant receptors (ORs), as both GPCR families are known to show strong basal activity. Consequently, ß2AR alone strongly enhanced the activity of HCN2 channels, and co-expression of ORs further diversified the HCN2 channel activity, which was totally abolished by an adenylate cyclase inhibitor. Thus, we conclude that the dual expression of constitutively active GPCRs establishes the diverse range of the basal cAMP pool size in resting cells through mutual additive or suppressive interactions, even in the absence of external stimulation.

Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors.

BACKGROUND: Neurons in the central nervous system (CNS) are generated by symmetric and asymmetric cell division of neural stem cells and their derivative progenitor cells. Cerebellar granule cells are the most abundant neurons in the CNS, and are generated by intensive cell division of granule cell precursors (GCPs) during postnatal development. Dysregulation of GCP cell cycle is causal for some subtypes of medulloblastoma. However, the details and mechanisms underlying neurogenesis from GCPs are not well understood. RESULTS: Using long-term live-cell imaging of proliferating GCPs transfected with a fluorescent newborn-granule cell marker, we found that GCPs underwent predominantly symmetric divisions, generating two GCPs or two neurons, while asymmetric divisions generating a GCP and a neuron were only occasionally observed, in both dissociated culture and within tissues of isolated cerebellar lobules. We found no significant difference in cell cycle length between proliferative and neurogenic divisions, or any consistent changes in cell cycle length during repeated proliferative division. CONCLUSIONS: Unlike neural stem cells in the cerebral cortex and spinal cord, which generate many neurons by repeated asymmetric division, cerebellar GCPs produce neurons predominantly by terminal symmetric division. These results indicate diverse mechanisms of neurogenesis in the mammalian brain.