Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.     

Transient receptor potential vanilloid 4-dependent calcium influx and ATP release in mouse and rat gastric epithelia

AIM: To explore the expression of transient receptor potential vanilloid 4 (TRPV4) and its physiological meaning in mouse and rat gastric epithelia.METHODS: RT-PCR and immunochemistry were used to detect TRPV4 mRNA and protein expression in mouse stomach and a rat normal gastric epithelial cell line (RGE1-01), while Ca²⁺-imaging and electrophysiology were used to evaluate TRPV4 channel activity. ATP release was measured by a luciferin-luciferase assay. Gastric emptying was also compared between WT and TRPV4 knockout mice.RESULTS: TRPV4 mRNA and protein were detected in mouse tissues and RGE1-01 cells. A TRPV4-specific agonist (GSK1016790A) increased intracellular Ca²⁺ concentrations and/or evoked TRPV4-like current activities in WT mouse gastric epithelial cells and RGE1-01 cells, but not TRPV4KO cells. GSK1016790A or mechanical stimuli induced ATP release from RGE1-01 cells while TRPV4 knockout mice displayed delayed gastric emptying in vivo.CONCLUSION: TRPV4 is expressed in mouse and rat gastric epithelium and contributes to ATP release and gastric emptying.     

瞬时受体电位超家族与胰岛素分泌

瞬时受体电位(TRP)通道蛋白由一系列具有不同离子通透性和门控特性的亚家族组成,在哺乳动物的多种组织和细胞中广泛表达.近年来大量研究发现,TRP通道蛋白在胰岛β细胞中广泛表达,通过多种机制参与胰岛素分泌的调节,并在糖尿病并发症的发生、发展中发挥重要作用.对TRP家族的研究将有助于深入了解糖尿病及其并发症的病理过程,从而为糖尿病的治疗提供新的药物靶点.     

Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans

Aims: The role of cannabinoid receptors in human islets of Langerhans has not been investigated in any detail, so the current study examined CB1 and CB2 receptor expression by human islets and the effects of pharmacological cannabinoid receptor agonists and antagonists on insulin secretion. Methods: Human islets were isolated from pancreases retrieved from heart‐beating organ donors. Messenger RNAs encoding human CB1 and CB2 receptors were amplified from human islet RNA by RT‐PCR and receptor localization within islets was identified by immunohistochemistry. Dynamic insulin secretion from human islets perifused with buffers supplemented with CB1 and CB2 receptor agonists and antagonists was quantified by radioimmunoassay. Results: RT‐PCR showed that both CB1 and CB2 receptors are expressed by human islets and immunohistochemistry indicated that receptor expression co‐localized with insulin‐expressing β‐cells. Perifusion experiments using isolated human islets showed that insulin secretion was reversibly stimulated by both CB1 and CB2 receptor agonists, with CB1 receptor activation associated with increased basal secretion whereas CB2 receptors were coupled to initiation and potentiation of insulin secretion. Antagonists at CB1 (N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) and CB2 (N‐(1,3‐Benzodioxol‐5‐ylmethyl)‐1,2‐dihydro‐7‐methoxy‐2‐oxo‐8‐(pentyloxy)‐3‐quinoline carboxamide) receptors failed to inhibit the stimulatory effects of the respective agonists and, unexpectedly, reversibly stimulated insulin secretion. Conclusions: These data confirm the expression of CB1 and CB2 receptors by human islets and indicate that both receptor subtypes are coupled to the stimulation of insulin secretion. They also implicate involvement of CB1/2 receptor‐independent pathways in the antagonist‐induced stimulatory effects.     

Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries

Local control of cerebral blood flow is regulated in part through myogenic constriction of resistance arteries. Although this response requires Ca2+ influx via voltage-dependent Ca2+ channels secondary to smooth muscle cell depolarization, the mechanisms responsible for alteration of vascular smooth muscle (VSM) cell membrane potential are not fully understood. A previous study from our laboratory demonstrated a critical role for a member of the transient receptor potential (TRP) superfamily of ion channels, TRPC6, in this response. Several other of the approximately 22 identified TRP proteins are also present in cerebral arteries, but their functions have not been elucidated. Two of these channels, TRPM4 and TRPM5, exhibit biophysical properties that are consistent with a role for control of membrane potential of excitable cells. We hypothesized that TRPM4/TRPM5-dependent currents contribute to myogenic vasoconstriction of cerebral arteries. Cation channels with unitary conductance, ion selectivity and Ca2+-dependence similar to those of cloned TRPM4 and TRPM5 were present in freshly isolated VSM cells. We found that TRPM4 mRNA was detected in both whole cerebral arteries and in isolated VSM cells whereas TRPM5 message was absent from cerebral artery myocytes. We also found that pressure-induced smooth muscle cell depolarization was attenuated in isolated cerebral arteries treated with TRPM4 antisense oligodeoxynucleotides to downregulate channel subunit expression. In agreement with these data, myogenic vasoconstriction of intact cerebral arteries administered TRPM4 antisense was attenuated compared with controls, whereas KCl-induced constriction did not differ between groups. We concluded that activation of TRPM4-dependent currents contributed to myogenic vasoconstriction of cerebral arteries.     

Nitric oxide inhibits prolactin secretion in pituitary cells downstream of voltage-gated calcium influx

The coupling between nitric oxide (NO)-cGMP signaling pathway and prolactin (PRL) release in pituitary lactotrophs has been established previously. However, the messenger that mediates the action of this signaling pathway on hormone secretion and the secretory mechanism affected, calcium dependent or independent, have not been identified. In cultured pituitary cells, basal PRL release was controlled by spontaneous voltage-gated calcium influx and was further enhanced by depolarization of cells and stimulation with TRH. Inhibition of constitutively expressed neuronal NO synthase decreased NO and cGMP levels and increased basal PRL release. The addition of a slowly releasable NO donor increased cGMP levels and inhibited basal PRL release in a time-dependent manner. Expression of inducible NO synthase also increased NO and cGMP levels and inhibited basal, depolarization-induced, and TRH-induced PRL release, whereas inhibition of this enzyme decreased NO and cGMP production and recovered PRL release. None of these treatments affected spontaneous and stimulated voltage-gated calcium influx. At basal NO levels, the addition of permeable cGMP analogs did not inhibit PRL secretion. At elevated NO levels, inhibition of cGMP production and facilitation of its degradation did not reverse inhibited PRL secretion. These experiments indicate that NO inhibits calcium-dependent PRL secretion in a cGMP-independent manner and downstream of voltage-gated calcium influx.     

Lidocaine inhibits prolactin secretion in GH4C1 cells by blocking calcium influx.

The mechanism of the inhibitory effect of local anesthetics on hormone secretion was studied in the GH4C1 line of rat pituitary tumor-derived cells. Lidocaine between 0.1 and 5 mM exerted significant dose-dependent inhibition on the increment in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion induced by 30 mM K+. For both effects the IC50 was 0.25 mM and maximal inhibition occurred at 5 mM. A normal response returned within 20 min after removal of lidocaine from the incubation medium. 1 microM tetrodotoxin had no effect on the 30 mM K+ induced [Ca2+]i transient or PRL secretion, indicating that Na+ channels are not involved in the inhibitory effect of lidocaine. Lidocaine similarly inhibited the [Ca2+]i increment and PRL secretion induced by 30% medium hyposmolarity and 1 microM Bay K 8644. Lidocaine was much less effective in inhibiting secretion induced by 1 microM phorbol 12-myristate 13-acetate (TPA) or 5 microM forskolin. 5 mM procaine produced effects similar to those of lidocaine. Our data suggest that in GH4C1 cells local anesthetics depress secretagogue-induced PRL secretion primarily by blocking Ca2+ influx, probably through L-type Ca2+ channels.     

P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.     

Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals

Olfactory sensory neurons (OSNs) in the main olfactory epithelium respond to environmental odorants. Recent studies reveal that these OSNs also respond to semiochemicals such as pheromones and that main olfactory input modulates animal reproduction, but the transduction mechanism for these chemosignals is not fully understood. Previously, we determined that responses to putative pheromones in the main olfactory system were reduced but not eliminated in mice defective for the canonical cAMP transduction pathway, and we suggested, on the basis of pharmacology, an involvement of phospholipase C. In the present study, we find that a downstream signaling component of the phospholipase C pathway, the transient receptor potential channel M5 (TRPM5), is coexpressed with the cyclic nucleotide-gated channel subunit A2 in a subset of mature OSNs. These neurons project axons primarily to the ventral olfactory bulb, where information from urine and other socially relevant signals is processed. We find that these chemosignals activate a subset of glomeruli targeted by TRPM5-expressing OSNs. Our data indicate that TRPM5-expressing OSNs that project axons to glomeruli in the ventral area of the main olfactory bulb are involved in processing of information from semiochemicals.     

Chylomicrons stimulate incretin secretion in mouse and human cells

Aims/hypothesis

Lipids are a potent stimulus for the secretion of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP). Traditionally, this effect was thought to involve the sensing of lipid digestion products by free fatty acid receptor 1 (FFA1) and G-protein coupled receptor 119 (GPR119) on the apical surface of enteroendocrine cells. However, recent evidence suggests that lipids may in fact be sensed basolaterally, and that fatty acid absorption and chylomicron synthesis may be a prerequisite for their stimulatory effect on gut peptide release. Therefore, we investigated the effect of chylomicrons on GLP-1 and GIP secretion in vitro.

Methods

The effect of chylomicrons on incretin secretion was investigated using GLUTag cells and duodenal cultures of both murine and human origin. The role of lipoprotein lipase (LPL) and FFA1 in GLUTag cells was assessed by pharmacological inhibition and small (short) interfering RNA (siRNA)-mediated knockdown. The effect of chylomicrons on intracellular calcium concentration ([Ca²⁺]_i) was determined by imaging GLUTag cells loaded with Fura-2. In the primary setting, the contributions of FFA1 and GPR119 were investigated using L cell-specific Gpr119 knockout cultures treated with the FFA1 antagonist GW1100.

Results

Chylomicrons stimulated GLP-1 release from GLUTag cells, and both GLP-1 and GIP secretion from human and murine duodenal cultures. Chylomicron-triggered GLP-1 secretion from GLUTag cells was largely abolished following lipase inhibition with orlistat or siRNA-mediated knockdown of Lpl. In GLUTag cells, both GW1100 and siRNA-mediated Ffar1 knockdown reduced GLP-1 secretion in response to chylomicrons, and, consistent with FFA1 G_q-coupling, chylomicrons triggered an increase in [Ca²⁺]_i. However, LPL and FFA1 inhibition had no significant effect on chylomicron-mediated incretin secretion in murine cultures. Furthermore, the loss of GPR119 had no impact on GLP-1 secretion in response to chylomicrons, even in the presence of GW1100.

Conclusions/interpretation

Chylomicrons stimulate incretin hormone secretion from GLUTag cells as well as from human and murine duodenal cultures. In GLUTag cells, the molecular pathway was found to involve LPL-mediated lipolysis, leading to the release of lipid species that activated FFA1 and elevated intracellular calcium.

     

Unique efficacy of Toll-like receptor 8 agonists in activating human neonatal antigen-presenting cells

Newborns are prone to microbial infection and have poor memory responses to multiple antigens. We have previously shown that human neonatal blood monocytes exhibit impaired TNF-alpha responses to most known TLR agonists, including the pure TLR7 agonist imiquimod. Surprisingly, however, neonatal TNF-alpha responses to the imiquimod congener R-848 (TLR 7/8) were fully intact. We now show that TLR8 agonists, including R-848 (TLR7/8), the imidazoquinoline congeners 3M-003 (TLR7/8) and 3M-002 (TLR8), as well as single-stranded viral RNAs (TLR8) induced robust production of the Th1-polarizing cytokines TNF-alpha and IL-12 from neonatal antigen-presenting cells (APCs) that substantially exceeds responses induced by TLR-2, -4, or -7 (alone) agonists. TLR8 agonists also effectively induced up-regulation of the costimulatory molecule CD40 on neonatal and adult myeloid dendritic cells (DCs). The strong activity of TLR8 agonists correlates with their induction of p38 MAP kinase phosphorylation and with degradation of IkappaB-alpha in both neonatal and adult monocytes. We conclude that TLR8 agonists are uniquely efficacious in activating costimulatory responses in neonatal APCs and suggest that these agents are promising candidate adjuvants for enhancing immune responses in human newborns.     

Insulin-like growth factor-1 increases intracellular calcium concentration in human primary neuroendocrine pancreatic tumor cells and a pancreatic neuroendocrine tumor cell line (BON-1) via R-type Ca2+ channels and regulates chromogranin a secretion in BON-1 cells

Insulin-like growth factor 1 (IGF-1) is a potent mitogenic and secretory factor that acts on voltage operated Ca(2+) channels (VOCCs). VOCCs are categorized into L-type channels (Ca(V)1.1-1.4), P/Q-type channels (Ca(V)2.1), N-type channels (Ca(V)2.2), R-type channels (Ca(V)2.3), and T-type channels (Ca(V)3.1-3.3). Aside from regulating membrane excitability, VOCCs influence chromogranin A (CgA) secretion in neuroendocrine tumor (NET) cells. It is not known, whether VOCCs play a role in the IGF-1-dependent regulation of CgA secretion in NET cells. We therefore studied the effects of IGF-1 on individual VOCC subtypes and characterized their role in mediating IGF-1-dependent regulation of CgA secretion in NET cells. Using specific modulators of VOCC subtypes, we identified the functional expression of L-, N-, P/Q- and R-type channels in primary as well as permanent models of NET. The IGF-1-induced intracellular Ca(2+) increase in NET cells was mainly due to the activation of R-type channel activity. The effects on intracellular calcium, observed in whole-cell patch-clamp recordings and fluorescence imaging, were partially blocked by the specific R-type channel blocker SNX-482 and antisense oligonucleotides against the alpha(1) subunit of this channel. IGF-1 potently induced CgA secretion. The effect of IGF-1 was reduced by both, inhibition of R-type channel activity and a reduction of R-type channel expression using antisense oligonucleotides. Since R-type channels exist in NET cells and couple to both, IGF-1 receptor signaling as well as CgA secretion, pharmacological interference with R-type channels may represent a new therapeutic option by blocking Ca(2+) signaling thereby abrogating IGF-1-dependent hypersecretion in NET disease.     

Transient receptor potential vanilloid 1-immunoreactive signals in murine enteric glial cells

AIM To investigate the possible involvement of transient receptor potential vanilloid 1(TRPV1) in maturation of enteric glial cells(EGCs).METHODS Immunohistochemical and immunocytochemical techniques were used to analyze EGC markers in myenteric plexus(MP) as well as cultured MP cells and EGCs using TRPV1 knockout(KO) mice.RESULTS We detected TRPV1-immunoreactive signals in EGC in the MP of wild-type(WT) but not KO mice. Expression of glial fibrillary acidic protein(GFAP) immunoreactive signals was lower at postnatal day(PD) 6 in KO mice, though the difference was not clear at PD 13 and PD 21. When MP cells were isolated and cultured from isolated longitudinal muscle-MP preparation from WT and KO mice, the yield of KO EGC was lower than that of WT EGC, while the yield of KO and WT smooth muscle cells showed no difference. Addition of BCTC, a TRPV1 antagonist, to enriched EGC culture resulted in a decrease in the protein ratio of GFAP to S100 B, another EGC/astrocyte-specific marker. CONCLUSION These results address the possibility that TRPV1 may be involved in the maturation of EGC, though further studies are necessary to validate this possibility.     

Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.     

8-Diethylamino-octyl-3,4,5-trimethoxybenzoate, a calcium store blocker, increases calcium influx, inhibits alpha-1 adrenergic receptor calcium mobilization, and alters iodide transport in FRTL-5 rat thyroid cells.

8-Diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8) is known to inhibit mobilization of calcium from intracellular stores but, more recently, other cellular effects have been described. In the present study, the effects of TMB-8 on cytosolic free calcium [Ca2+]i levels in FRTL-5 rat thyroid cells were determined using the fluorescent dye, Indo-1. TMB-8 increased [Ca2+]i in a dose-dependent manner, with a maximum rise from 120 +/- 7 nM to 229 +/- 16 nM (90 +/- 5% increase) at 5 x 10(-4) M. This effect was considerably reduced in Ca2+ free buffer, demonstrating a dependency upon extracellular calcium influx but not upon membrane potential and which did not involve the Na+/Ca2+ exchanger. In Ca2+ free buffer TMB-8, at a dose which did not affect [Ca2+]i, completely prevented norepinephrine (10(-5) M) from mobilizing intracellular Ca2+. To determine whether TMB-8 affected differentiated functions, iodide uptake and efflux studies were performed with 125I. TMB-8 (10(-4) M) inhibited iodide uptake by approximately 40% without affecting efflux. At 10(-3) M TMB-8, efflux was also enhanced. These studies demonstrate that TMB-8 has at least two effects on [Ca2+]i, promoting calcium influx and preventing alpha-1 adrenergic mobilization from intracellular stores. TMB-8 also has multiple effects on 125I transport, both inhibiting influx and increasing efflux. The results emphasize the importance of characterizing the behavior of this compound in any cell system before using it as a biological probe.