Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.     

Studies with GIP/Ins cells indicate secretion by gut K cells is KATP channel independent

K cells are a subpopulation of enteroendocrine cells that secrete glucose-dependent insulinotropic polypeptide (GIP), a hormone that promotes glucose homeostasis and obesity. Therefore, it is important to understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell lines secreted hormones in response to many GIP secretagogues except glucose. In contrast, glyceraldehyde and methyl pyruvate stimulated hormone release. Measurements of intracellular glucose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate levels, as well as glycolytic flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was not impaired. Analogous results were obtained using glucose-responsive MIN6 insulinoma cells. Citrate levels increased similarly in glucose-treated MIN6 and GIP/Ins cells. Thus pyruvate entered the tricarboxylic acid cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP-to-ADP ratio in GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than increased, ATP/ADP. Thus nutrient-regulated secretion is independent of the ATP-dependent potassium (K(ATP)) channel. Antibody staining of mouse intestine demonstrated that enteroendocrine cells producing GIP, glucagon-like peptide-1, CCK, or somatostatin do not express detectable levels of inwardly rectifying potassium (Kir) 6.1 or Kir 6.2, indicating that release of these hormones in vivo may also be K(ATP) channel independent. Conversely, nearly all cells expressing chromogranin A or substance P and approximately 50% of the cells expressing secretin or serotonin exhibited Kir 6.2 staining. Compounds that activate calcium mobilization were potent secretagogues for GIP/Ins cells. Secretion was only partially inhibited by verapamil, suggesting that calcium mobilization from intracellular and extracellular sources, independent from K(ATP) channels, regulates secretion from some, but not all, subpopulations of enteroendocrine cells.     

The predominant role of IP₃ type 1 receptors in activation of store-operated Ca²+ entry in liver cells

Physiologically, hormone induced release of Ca2+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP?) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP? receptors act as channels, releasing Ca2+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca2+ stores leads to activation of store-operated Ca2+ channels on the plasma membrane which replenishes lost Ca2+ and sustain Ca2+ signalling. There are three isoforms of IP? receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca2+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca2+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca2+ channels, and Ca2+ release activated Ca2+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP? receptor but not the others. Here we show that H4IIE liver cells express all three types of IP? receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca2+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca2+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP? receptors.     

Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes

Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+-signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+-signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signalling.     

Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt20 cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt20 cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2+ release from ER by IP3Rs within cells.     

Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures

Fast nongenomic steroid actions in several cell types seem to be mediated by second messengers such as intracellular calcium ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)). We have shown the presence of both slow calcium transients and IP(3) receptors associated with cell nuclei in cultured skeletal muscle cells. The effect of steroids on [Ca(2+)](i) was monitored in Fluo 3-acetoxymethyl ester-loaded myotubes by either confocal microscopy or fluorescence microscopy, with the use of out-of-focus fluorescence elimination. The mass of IP(3) was determined by radioreceptor displacement assay. [Ca(2+)](i) changes after either aldosterone (10-100 nM) or testosterone (50-100 nM) were observed; a relatively fast (<2 min) calcium transient, frequently accompanied by oscillations, was evident with both hormones. A slow rise in [Ca(2+)](i) that reached its maximum after a 30-min exposure to aldosterone was also observed. Calcium responses seem to be fairly specific for aldosterone and testosterone, because several other steroid hormones do not induce detectable changes in fluorescence, even at 100-fold higher concentrations. The mass of IP(3) increased transiently to reach two- to threefold the basal level 45 s after addition of either aldosterone or testosterone, and the IP(3) transient was more rapid than the fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor, or cyproterone acetate, an inhibitor of the testosterone receptor, had no effect on the fast [Ca(2+)](i) signal or in the increase in IP(3) mass. These signals could mean that there are distinct nongenomic pathways for the action of these two steroids in skeletal muscle cells.     

Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes

Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non- muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation- contraction coupling in the heart.     

Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins

A number of previous reports have suggested that inositol 1,4, 5-trisphosphate receptors (IP(3)Rs) are present in the plasma membranes of cells. We confirm this directly in the present study by demonstrating that a significant proportion of the IP(3)Rs found in A431 cells, Jurkat cells, and rat parotid acini can be biotinylated by the extracellular application of sulfo-N-hydroxysuccinimide-biotin to intact cells. This labeling cannot be accounted for by the reaction of sulfo-N-hydroxysuccinimide-biotin with intracellular IP(3)Rs since calnexin and the SERCA2 ATPase, both integral membrane proteins of the endoplasmic reticulum, are not labeled under the same experimental conditions. Individual IP(3)R subtypes were detected using subtype-specific antibodies. A431 cells expressed only the type-3 IP(3)R, and 23% of this protein was in the biotinylated (plasma membrane) fraction. Jurkat cells and rat parotid cells expressed all three IP(3)R subtypes. Contrary to earlier results suggesting that only the type-3 IP(3)R might localize to the plasma membrane, we found that significant amounts (5-14%) of all three subtypes could be identified in the biotinylated fractions of Jurkat and rat parotid cells. Our results suggest a role for IP(3)Rs in plasma membrane as well as intracellular membrane function.     

Bombesin and nutrients independently and additively regulate hormone release from GIP/Ins cells

Glucose-dependent insulinotropic polypeptide (GIP) regulates glucose homeostasis and high-fat diet-induced obesity and insulin resistance. Therefore, elucidating the mechanisms that regulate GIP release is important. GIP is produced by K cells, a specific subtype of small intestinal enteroendocrine (EE) cell. Bombesin-like peptides produced by enteric neurons and luminal nutrients stimulate GIP release in vivo. We previously showed that PMA, bombesin, meat hydrolysate, glyceraldehyde, and methylpyruvate increase hormone release from a GIP-producing EE cell line (GIP/Ins cells). Here we demonstrate that bombesin and nutrients additively stimulate hormone release from GIP/Ins cells. In various cell systems, bombesin and PMA regulate cell physiology by activating PKD signaling in a PKC-dependent fashion, whereas nutrients regulate cell physiology by inhibiting AMPK signaling. Western blot analyses of GIP/Ins cells using antibodies specific for activated and/or phosphorylated forms of PKD and AMPK and one substrate for each kinase revealed that bombesin and PMA, but not nutrients, activated PKC, but not PKD. Conversely, nutrients, but not bombesin or PMA, inhibited AMPK activity. Pharmacological studies showed that PKC inhibition blocked bombesin- and PMA-stimulated hormone release, but AMPK activation failed to suppress nutrient-stimulated hormone secretion. Forced expression of constitutively active vs. dominant negative PKDs or AMPKs failed to perturb bombesin- or nutrient-stimulated hormone release. Thus, in GIP/Ins cells, PKC regulates bombesin-stimulated hormone release, whereas nutrients may control hormone release by regulating the activity of AMPK-related kinases, rather than AMPK itself. These results strongly suggest that K cells in vivo independently respond to neuronal vs. nutritional stimuli via two distinct signaling pathways.     

Repeated immobilization stress reduces the gene expression of the type 1 and 2 IP3 receptors in stellate ganglia

Inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by phosphoinositid hydrolysis and triggers IP(3) receptor (IP(3)R) mediated calcium release from intracellular pools. To determine whether immobilization stress affects the gene expression and protein level of IP(3)R in stellate ganglia, animals were immobilized once for 2h and/or for 7 days, 2h daily. After decapitation, stellate ganglia were extirpated and the gene expression of IP(3) receptors was evaluated. Protein levels of IP(3) receptor were measured by Western blot analysis using the antibody against IP(3) receptor. In the present work, we clearly show that type 1 and 2 IP(3) receptors, but not the type 3 IP(3) receptor, are expressed in stellate ganglia. Both types, type 1 and 2 IP(3) receptors, are not significantly affected by single 2h immobilization stress on mRNA and protein level. However, gene expression of both these types is significantly reduced by repeated immobilization stress for 7 days, 2h daily. The IP(3) receptor protein is reduced as well. Physiological relevance of our observations remains to be elucidated.     

Stimulation of the P2Y Purinergic Receptor on Type 1 Astroglia Results in Inositol Phosphate Formation and Calcium Mobilization

Cultured astroglia express purinergic receptors that initiate phosphoinositide metabolism and calcium mobilization. Experiments were conducted to characterize the purinergic receptor subtype on type 1 astroglia responsible for stimulation these second-messenger systems. Inositol phosphate (IP) accumulation and calcium mobilization were measured after stimulation with ATP or purinergic receptor subtype-selective ATP analogues. ATP (10(-5) M) increased IP accumulation severalfold. Dose-effect assays monitoring astroglial IP accumulation revealed the order of potency that defines the P2Y receptor: 2-methylthioadenosine 5'-triphosphate greater than ATP greater than alpha beta-methyleneadenosine 5'-triphosphate greater than beta gamma-methyleneadenosine 5'-triphosphate. The influence of ATP on intracellular calcium levels in individual type 1 astroglia was examined using the calcium indicator dye, fura-2. Dose-effect experiments indicated that ATP was equally potent for generating inositol phosphates and increasing cellular calcium. The most prevalent response (87% of total responses) to ATP consisted of a rapid increase in calcium to a peak level that was approximately five times greater than the prestimulation level. This peak was followed by a decline to a plateau level that was significantly above baseline. This plateau phase of the calcium increase was maintained for at least 5 min in the presence of ATP and was dependent on external calcium. Many (23%) astroglia exhibited spontaneous calcium oscillations whose frequency and magnitude increased after the addition of 10(-5) M ATP. Immunocytochemical staining indicated that the responses occurred in glial fibrillary acidic protein positive cells. We conclude that type 1 astroglia express the P2Y purinergic receptor which regulates IP production and calcium mobilization.     

ERK binds, phosphorylates InsP3 type 1 receptor and regulates intracellular calcium dynamics in DT40 cells

Modulation on the duration of intracellular Ca(2+) transients is essential for B-cell activation. We have previously shown that extracellular-signal-regulated kinase (ERK) can phosphorylate inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) at serine 436 and regulate its calcium channel activity. Here we investigate the potential physiological interaction between ERK and IP(3)R1 using chicken DT40 B-cell line in which different mutants are expressed. The interaction between ERK and IP(3)R1 is confirmed by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. This constitutive interaction is independent of either ERK kinase activation or IP(3)R1 phosphorylation status. Back phosphorylation analysis further shows that type 1 IP(3)R (IP(3)R1) is phosphorylated by ERK in anti-IgM-activated DT40 cells. Finally, our data show that the phosphorylation of Ser 436 in the IP(3)-binding domain of IP(3)R1 leads to less Ca(2+) release from endoplasmic reticulum (ER) microsomes and accelerates the declining of calcium increase in DT40 cells in response to anti-IgM stimulation.     

Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.     

Role of the inositol 1,4,5-trisphosphate receptor in Ca(2+) feedback inhibition of calcium release-activated calcium current (I(crac)).

We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) is re-accumulated at sites that regulate I(crac). Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.     

Possible role of PEPT1 in gastrointestinal hormone secretion

Oligopeptides originating from ingested meal stimulate the secretion of various gastrointestinal hormones, but the mechanism is unknown. In this study, we show that transfection of oligopeptide transporter 1 (PEPT1) in STC-1 cells, a murine enteroendocrine cell line, evokes di-peptide-stimulated hormone secretion in a pH-dependent manner. Measurement of membrane potentials shows that PEPT1- transfected STC-1 cells are depolarized by di-peptide glycyl-glycine but not by glycine monomer. Glycyl-glycine stimulation induces a rise in the intracellular calcium concentration in PEPT1-transfected STC-1 cells. The secretion induced by glycyl-glycine in PEPT1-transfected STC-1 cells was blocked by nifedipine, a Ca(2+) channel blocker, suggesting that the secretion is triggered by Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. These data suggest that PEPT1 mediates oligopeptide-induced hormone secretion in enteroendocrine cells.