Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca(2+).

BACKGROUND: Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures. METHODS: Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm. RESULTS: U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2. CONCLUSIONS: In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.     

Expression of gastrin-releasing peptide receptors in endometrial cancer

BACKGROUND: The Bombesin (BBS)-related peptide, gastrin-releasing peptide, and its cognate receptor are ectopically expressed by many cancers, in which they regulate tumor proliferation and metastasis. But, their role in endometrial cancers is unknown. The purpose of this study was to determine whether endometrial cancer cell lines express functional BBS receptors and to determine whether they were coupled to the regulation of vascular endothelial growth factor (VEGF-A) expression. STUDY DESIGN: Endometrial cancer cell lines (HEC-1A, KLE, and AN3CA) were cultured according to the recommendations of the American Tissue Culture Collection. Ishikawa cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Before BBS treatment, all cell lines were placed in serum-free, phenol-free media for 24 hours. BBS-stimulated increases in intracellular Ca(2+) ([Ca(2+)]i) were used to assess functional BBS receptor status. VEGF-A mRNA expression was determined by Northern blotting. RESULTS: BBS (100 nM) stimulated an increase in [Ca(2+)]i in HEC-1A, Ishikawa, and KLE cells, indicating the presence of functional BBS receptors. This increase did not occur in AN3CA cells. BBS stimulated a time-dependent increase in VEGF-A mRNA expression in Ishikawa and KLE cells. Ishikawa cells exhibited a peak of VEGF-A mRNA expression between 8 and 12 hours with a partial decline by 24 hours. KLE cells showed a relatively small increase at 12 hours. In contrast, HEC-1A cells exhibited a high baseline level of VEGF-A mRNA expression and did not show a response to BBS. CONCLUSIONS: These data demonstrate that endometrial cancer cell lines express functional BBS receptors. In Ishikawa, KLE, and HEC-1A cells, BBS receptors are coupled to the regulation of VEGF-A mRNA expression.     

Gastrointestinal peptide signalling in health and disease.

Gastrointestinal peptides including mammalian bombesin-like peptides, cholecystokinin (CCK), gastrin, and neurotensin stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a number of fundamental processes including development, inflammation, tissue regeneration, and neoplastic transformation. These agonists bind to G protein-coupled receptors (GPCRs) that promote Galpha q-mediated activation of beta isoforms of phospholipase C to produce two second messengers: Inositol (1,4,5) trisphosphate {Ins (1, 4, 5) P3} that mobilises Ca2+ from internal stores, and diacylglycerol that activates the classic and new isoforms of the protein kinase C (PKC) family. PKCs play a critical part in transducing bombesin/gastrin releasing peptide (GRP) receptor signals into activation of protein kinase cascades. Protein kinase D (PKD), a serine/threonine protein kinase with distinct structural and enzymological properties, is activated by phosphorylation in living cells through a new PKC-dependent signal transduction pathway. GPCR agonists including bombesin/GRP induce a rapid and striking activation of PKD by PKC. These results indicate that PKD functions downstream from PKCs and identify a new phosphorylation cascade that is activated by gastrointestinal peptide agonists. The bombesin/GRP GPCR also promotes rapid Rho-dependent assembly of focal adhesions, formation of actin stress fibres and tyrosine phosphorylation of multiple cellular proteins. We identified p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (CAS) and paxillin as prominent targets of gastrointestinal peptide-stimulated tyrosine phosphorylation and developed a model that envisages a G12/Rho-dependent pathway connecting GPCR activation to the tyrosine phosphorylation of these focal adhesion proteins. Separate pathways mediate gastrointestinal peptide stimulation of additional tyrosine kinase pathways including transactivation of Src and epidermal growth factor receptor (EGFR). Tyrosine phosphorylation has a critical role in gastrointestinal peptide-induced cellular migration and cooperates with Gq-stimulated events to promote mitogenesis. The growth-promoting effects of neuropeptides and the elucidation of the signalling pathways that mediate their effects assume an added importance because these agonists and their receptors are increasingly implicated in sustaining the proliferation of clinically aggressive solid tumours including those from lung, pancreas, and colon.     

Protein kinase G inhibits flow-induced Ca2+ entry into collecting duct cells

The renal cortical collecting duct (CCD) contributes to the maintenance of K(+) homeostasis by modulating renal K(+) secretion. Cytosolic Ca(2+) ([Ca(2+)](i)) mediates flow-induced K(+) secretion in the CCD, but the mechanisms regulating flow-induced Ca(2+) entry into renal epithelial cells are not well understood. Here, we found that atrial natriuretic peptide, nitric oxide, and cyclic guanosine monophosphate (cGMP) act through protein kinase G (PKG) to inhibit flow-induced increases in [Ca(2+)](i) in M1-CCD cells. Coimmunoprecipitation, double immunostaining, and functional studies identified heteromeric TRPV4-P2 channels as the mediators of flow-induced Ca(2+) entry into M1-CCD cells and HEK293 cells that were coexpressed with both TRPV4 and TRPP2. In these HEK293 cells, introducing point mutations at two putative PKG phosphorylation sites on TRPP2 abolished the ability of cGMP to inhibit flow-induced Ca(2+) entry. In addition, treating M1-CCD cells with fusion peptides that compete with the endogenous PKG phosphorylation sites on TRPP2 also abolished the cGMP-mediated inhibition of the flow-induced Ca(2+) entry. Taken together, these data suggest that heteromeric TRPV4-P2 channels mediate the flow-induced entry of Ca(2+) into collecting duct cells. Furthermore, substances such as atrial natriuretic peptide and nitric oxide, which increase cGMP, abrogate flow-induced Ca(2+) entry through PKG-mediated inhibition of these channels.     

Gut peptide receptor expression in human pancreatic cancers

OBJECTIVE: To determine the prevalence of gastrointestinal (GI) peptide receptor expression in pancreatic cancers, and to further assess signaling mechanisms regulating neurotensin (NT)-mediated pancreatic cancer growth. SUMMARY BACKGROUND DATA: Pancreatic cancer remains one of the leading causes of GI cancer death; novel strategies for the early detection and treatment of these cancers is required. Previously, the authors have shown that NT, an important GI hormone, stimulates the proliferation of an NT receptor (NTR)-positive pancreatic cancer. METHODS: A total of 26 human pancreatic adenocarcinomas, obtained after resection, and 5 pancreatic cancer xenografts were analyzed for expression of NTR, vasoactive intestinal peptide receptor (VIPR), substance P receptor (SPR), and gastrin-releasing peptide receptor (GRPR). In addition, NTR expression, [Ca2+]i mobilization, and growth in response to NT was determined in L3.6, a metastatic pancreatic cancer cell line. RESULTS: Neurotensin receptor was expressed in 88% of the surgical specimens examined and all five of the pancreatic cancer xenografts. In contrast, VIPR, SPR, and GRPR expression was detected in 31%, 27%, and 8% of pancreatic cancers examined, respectively. Expression of NTR, functionally coupled to the Ca2+ signaling pathway, was identified in L3.6 cells; treatment with NT (10 micromol/L) stimulated proliferation of these cells. CONCLUSIONS: The authors demonstrated NTR expression in most of the pancreatic adenocarcinomas examined. In contrast, VIPR, SPR, and GRPR expression was detected in fewer of the pancreatic cancers. The expression of NTR and other peptide receptors suggests the potential role of endocrine manipulation in the treatment of these cancers. Further, the presence of GI receptors may provide for targeted chemotherapy or radiation therapy or in vivo scintigraphy for early detection.     

Roles of gastrointestinal hormones in pancreatic cancer

Several gastrointestinal (GI) hormones, such as gastrin, cholecystokinin, and bombesin, have been reported to affect the development of pancreatic cancer. The receptors for these hormones are found in normal and neoplastic pancreatic cells. Activation of these receptors enhances pancreatic carcinogenesis and promotes the growth of established pancreatic carcinoma either in vitro or in vivo. On the other hand, some studies have shown that these GI hormones may have no effect or may play an inhibitory role in the development of pancreatic cancer. The reasons for the apparent discrepancies in the published literature are discussed in this review. In recent years, increasing emphasis has been placed on the effects of GI hormones on cancer invasion and metastasis. As the transition from noninvasion to the invasive state is the crucial event in cancer development, further investigation of the way in which GI hormones affect the invasion and metastasis of pancreatic cancer may be important for the development of new therapeutic approaches with eventual clinical utility. Received for publication on Oct. 23, 1999; accepted on Feb. 2, 2000     

Interaction of intravenous anesthetics with recombinant human M1-M3 muscarinic receptors expressed in chinese hamster ovary cells

Previous reports suggest that the effects of propofol, ketamine, and thiopental on airway tone may be because of modulation of parasympathetic activity. We examined if these anesthetics interact with recombinant human M1-M3 muscarinic receptors expressed in Chinese hamster ovary cells (CHO-M1, M2, and M3) using the displacement of 0.4 nM of l-[N-methyl-(3)H]scopolamine methyl chloride([(3)H]NMS). In addition, functional studies were performed by fluorometrically monitoring methacholine (1 mM) stimulated intracellular Ca(2+) ([Ca(2+)](i)) responses. Ketamine concentration dependently displaced [(3)H]NMS binding to CHO-M1, M2, and M3 cells with affinity, pK(i) (mean K(i)) values of 4.34 +/- 0.14 (45 micro M), 3.53 +/- 0.10 (294 micro M), and 3.61 +/- 0.02 (246 micro M), respectively. The effects at M1 were in the clinical range. Ketamine did not affect either basal or methacholine stimulated increase in [Ca(2+)](i) in CHO-M1 cells. Thiopental significantly displaced [(3)H]NMS binding to M3 (pKi [mean Ki] = 4.12 +/- 0.06 [75 micro M]) but not M1 or M2 receptors. Thiopental (10(-5)-10(-3) M) concentration dependently inhibited methacholine stimulated increase in [Ca(2+)](i) in CHO-M3 cells. Propofol and barbituric acid did not interact with any muscarinic receptor subtype. We suggest that at the level of [Ca(2+)](I), thiopental may possess M3 antagonist activity, whereas there are no functional consequences of the interaction of ketamine with the M1 receptor. IMPLICATIONS: In this study using recombinant human M1-M3 muscarinic receptors, we show that for agonist-stimulated increases in intracellular Ca(2+) thiopental acts as a M3 antagonist.     

Mechanism of estrogens-induced increases in intracellular Ca(2+) in PC3 human prostate cancer cells

BACKGROUND: The effect of estrogens (diethylstilbestrol [DES], 17 beta-estradiol) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in hormone-insensitive PC3 human prostate cancer cells was examined. METHODS: [Ca(2+)](i) changes in suspended cells were measured by using the Ca(2+)-sensitive fluorescent dye fura-2. RESULTS: Estrogens (1--20 microM) increased [Ca(2+)](i) concentration-dependently with DES being more potent. Ca(2+) removal inhibited 50 +/- 10% of the signal. In Ca(2+)-free medium, pretreatment with 20 microM estrogens abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), but pretreatment with CCCP and thapsigargin did not alter DES-induced Ca(2+) release and partly inhibited 17 beta-estradiol-induced Ca(2+) release. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 1- 20 microM estrogens in Ca(2+)-free medium. Pretreatment with 1 microM U73122 to block phospholipase C-coupled inositol 1,4,5-trisphosphate formation did not alter estrogens-induced Ca(2+) release. The effect of 20 microM estrogen on [Ca(2+)](i) was not affected by pretreatment with 0.1 microM estrogens. CONCLUSIONS: Estrogen induced significant Ca(2+) release and Ca(2+) influx in an inositol 1,4,5-trisphosphate-independent manner in PC3 cells. These effects of estrogens on Ca(2+) signaling appear to be nongenomic. Prostate 47:141-148, 2001.     

Gastrointestinal hormones and cell proliferation

There is no question that gut peptides are trophic for normal gut mucosa. Gut peptides can function in an endocrine, paracrine or autocrine fashion. We examined th effects of gut peptides on the growth of animal and human cancers of the gastrointestinal (GI) tract and pancreas in vivo and in vitro. We also examined the role of growth factors and bioamines in the regulation of growth of human endocrine tumors. Our studies have shown that gut peptides (gastrin, VIP, neurotensin, and bombesin) regulate growth of some cancers of the GI tract and pancreas. We have found that peptide action is mediated through specific receptors and that cell-specific differences in receptor expression occur. We have also begun to examine the intracellular signal-transduction pathways involved in endocrine and autocrine actions of these peptides on growth of GI cancers. We have found that cell type-specific differences exist among the various signal transduction pathways (cyclic AMP, phosphatidylinositol hydrolysis (PI), intracellular calcium ([Ca²⁺]_i) mobilization and tyrosine phosphorylation) and that different receptors for the same hormone may be linked to different signal transduction pathways depending upon cell type. We have also found that autocrine growth regulation of human pan creatic carcinoid occurs through specific receptor-mediated( signal-transduction pathways. We will discuss the mechanisms of action and potential therapeutic uses of manipulation o: gut hormone levels or hormone antagonists to inhibit the growth of GI tract cancers.     

Presence of receptors for bombesin/gastrin-releasing peptide and mRNA for three receptor subtypes in human prostate cancers

BACKGROUND: Bombesin-like peptides can function as autocrine or paracrine growth factors and stimulate the growth of some cancer cells, including human prostate cancer. Three bombesin receptor subtypes, termed gastrin-releasing peptide receptor (GRPR), neuromedin B receptor (NMBR), and bombesin receptor subtype 3 (BRS-3), have been identified in rodents and humans. METHODS: We investigated the presence and characteristics of the functional receptors for bombesin/GRP in human prostate adenocarcinoma specimens by radio-receptor assay and the mRNA expression of the three bombesin receptor subtypes by RT-PCR. RESULTS: Of the 80 specimens of primary prostate cancer examined by receptor binding assays, 50 ( approximately 63%) showed high-affinity, low-capacity binding sites for bombesin/GRP, and 12 of these 50 receptor-positive specimens also showed a second binding site. Of the 22 prostate cancer specimens analyzed by RT-PCR, 20 (91%) expressed GRPR mRNA, 3 (14%) showed NMBR mRNA, and 2 ( approximately 9%) revealed BRS-3 mRNA. No correlation was observed between receptor expression and patients' age or pathological data. CONCLUSIONS: The detection of a wide distribution of bombesin/GRP receptors in human prostate carcinomas supports the view that they may be involved in modulation of tumor progression and suggests that approaches based on binding of bombesin receptor antagonists or new targeted cytotoxic bombesin analogs to prostate cancers could be considered for the therapy.     

Effect of neurokinins on canine prostate cell physiology

BACKGROUND: Sensory peptide neurotransmitters have been implicated as significant regulators of prostate growth. This study was designed to evaluate the role of neurokinins in proliferation, differentiation, and contraction of canine prostate cells in culture. METHODS: NK1, NK2, and NK3 receptor subtypes were localized in canine prostate tissue by immunocytochemistry and ligand binding studies. Functional effects of neurokinin agonists were tested on cell differentiation (expression of smooth muscle actin (SMA)), proliferation (MTS assay), and contraction of canine prostate cells in culture. RESULTS: Immunocytochemical staining of canine prostate sections revealed strong stromal staining for NK1 together with weak stromal staining for NK2 and even weaker staining for NK3. Furthermore, there was overlapping localization of NK1 receptors, substance P (SP), and calcitonin gene-regulated peptide (CGRP) in prostate tissue sections. SP caused concentration-dependent increase in SMA expression that was attenuated in a concentration-dependent manner by YM-44778, a non-selective antagonist for neurokinin receptors, but not by either the NK2 antagonist (SR-48968) nor by the NK3 antagonist (SB-223412). SP and neurokinin A (NKA) also caused a modest contraction of stromal cells in collagen gels. NKA stimulated proliferation of prostate epithelial cells without any apoptotic effect, which was attenuated by SR-48968. Surprisingly, in binding studies NK3 appeared to be the most abundant neurokinin receptor subtype, although functional studies failed to reveal significant coupling of this receptor. CONCLUSIONS: Our results suggest that, at least in vitro, neurokinins have modest effects on canine prostate epithelial cell proliferation, stromal differentiation, and contraction.     

Effect of high glucose on basal intracellular calcium regulation in rat mesangial cell

BACKGROUND: A number of cellular mechanisms are critically dependent on intracellular Ca(2+) homeostasis. A sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) is capable of activating a number of potentially harmful processes including phenotype change to secretory type, dysregulated cell proliferation, and cell injury and death. Mesangial cells (MCs) play an important role in the pathophysiology of diabetic nephropathy. METHODS: We evaluated the effect of high glucose on basal [Ca(2+)](i) in the unstimulated state and identified its contributing pathways. MCs were isolated and cultured from Sprague-Dawley rats. [Ca(2+)](i) was measured by fluorometric technique with fura-2AM. RESULTS: In a dose-dependent manner, superfusion of MCs with Tyrode's solution containing high glucose (30 and 50 mM) induced a delayed spontaneous increase in [Ca(2+)](i), which was not found in those with normal (5.5 mM) glucose or mannitol. The high glucose-induced increase in [Ca(2+)](i)()occurred through transmembrane influx of extracellular Ca(2+) and was blocked by SKF96365, an inhibitor of store-operated Ca(2+) influx. Na(+)-Ca(2+) exchanger (NCX) activity, a major channel regulating basal [Ca(2+)](i), and the clearing ability of intracellular Ca(2+) were depressed after MCs were cultured in high-glucose medium. Western blot analysis revealed the decreased expression of a 70-kD NCX protein in MCs cultured in high-glucose medium. CONCLUSIONS: A high-glucose concentration induced a spontaneous increase in basal [Ca(2+)](i) of MCs without stimulation. There was a decrease in the activity of NCX in the high-glucose condition, which seems to occur at the level of protein expression. The present results provide a novel insight into the mechanisms of diabetic nephropathy in that intracellular Ca(2+) homeostasis is an important secondary messenger and a mediator in hormonal signaling.     

K+ channel currents in rat ventral prostate epithelial cells

BACKGROUND: Electrophysiological function of the normal prostate has not been extensively studied. In particular, ion channel currents and their regulation have not been studied in freshly-isolated prostate cells. METHODS: Rat prostate secretory epithelial (RPSE) cells were isolated by collagenase treatment. Columnar epithelial cells were used for nystatin-perforated, whole-cell voltage clamp, and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. RESULTS: Step-like depolarizing pulses (900 msec) starting from - 90 mV induced outwardly rectifying K(+) currents without inactivation. ACh (10 microM) or ATP (100 microM) increased the outward current and hyperpolarized the cell membrane potential. Ionomycin (0.1 microM), a Ca(2+) ionophore, induced a similar increase in the outward current. TEA (5 mM), charybdotoxin (50 nM), and iberiotoxin (30 nM) inhibited the effect of ACh (or ATP) on the outward current, whereas apamin (100 nM) had no effect. The [Ca(2+)](i) of RPSE cells was increased by ACh, ATP, and UTP. CONCLUSIONS: RPSE cells have iberiotoxin-sensitive Ca(2+)-activated K(+) channels that may play an important role in the exocrine secretions of the prostate.     

Dynamic imaging of endoplasmic reticulum Ca2+ concentration in insulin-secreting MIN6 Cells using recombinant targeted cameleons: roles of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 and ryanodine receptors

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](cyt), and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse beta-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.     

Flow-induced [Ca2+]i increase depends on nucleotide release and subsequent purinergic signaling in the intact nephron

Flow induces cytosolic Ca(2+) increases ([Ca(2+)](i)) in intact renal tubules, but the mechanism is elusive. Mechanical stimulation in general is known to promote release of nucleotides (ATP/UTP) and trigger auto- and paracrine activation of P2 receptors in renal epithelia. It was hypothesized that the flow-induced [Ca(2+)](i) response in the renal tubule involves mechanically stimulated nucleotide release. This study investigated (1) the expression of P2 receptors in mouse medullary thick ascending limb (mTAL) using P2Y(2) receptor knockout (KO) mice, (2) whether flow increases induce [Ca(2+)](i) elevations in mTAL, and (3) whether this flow response is affected in mice that are deplete of the main purinergic receptor. [Ca(2+)](i) was imaged in perfused mTAL with fura-2 or fluo-4. It is shown that luminal and basolateral P2Y(2) receptors are the main purinergic receptor in this segment. Moreover, the data suggest presence of basolateral P2X receptors. Increases of tubular flow were imposed by promptly rising the inflow pressure, which triggered a marked increase of [Ca(2+)](i). This [Ca(2+)](i) response was significantly reduced in P2Y(2) receptor KO tubules (fura-2 ratio increase WT 0.44 +/- 0.09 [n = 28] versus KO 0.16 +/- 0.04 [n = 13]). Furthermore, the flow response was greatly inhibited with luminal and basolateral scavenging of extracellular ATP (apyrase 7.5 U/ml) or blockage of P2 receptors (suramin 300 microM). The flow response could still be elicited in the absence of extracellular Ca(2+). These results strongly suggest that increase of tubular flow elevates [Ca(2+)](i) in intact renal epithelia. This flow response is caused by release of bilateral nucleotides and subsequent activation of P2 receptors.     

Modulation of angiotensin II-mediated signalling by heparan sulphate glycosaminoglycans.

BACKGROUND: Heparin and angiotensin-converting enzyme inhibitors can be used as a therapeutic option in diabetic nephropathy (DN). Although the mode of action is poorly understood, both agents may retard the progression of DN. Previously, we demonstrated that angiotensin II (Ang II) has an inhibitory effect on the production of heparan sulphate proteoglycan (HSPG) in mesangial cells (MCs). We have now studied the influence of heparin on the Ang II-induced intracellular Ca(2+) release and activation of nuclear factor kappa B (NF-kappaB). METHODS: Human MCs were isolated from renal cortex and cultivated to measure Ca(2+) influx and NF-kappaB activation. RESULTS: Stimulation of MCs with 100 nM Ang II resulted in a rapid increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), followed by a decline to baseline level. The addition of heparin resulted in an oscillatory pattern of Ca(2+) influxes upon Ang II stimulation. Whereas the rapid increase in [Ca(2+)](i) was most likely due to release from intracellular stores, oscillations in [Ca(2+)](i) were dependent on the presence of extracellular Ca(2+). Heparin alone did not induce Ca(2+) influx. Both the initial increase and the subsequent oscillations in [Ca(2+)](i) could be blocked by losartan. In MCs with chemically or enzymatically altered membrane-associated heparan sulphate glycosaminoglycan (HS-GAG), Ang II stimulation resulted in [Ca(2+)](i) oscillations. Interestingly, in these cells, the addition of heparin or GAG completely prevented [Ca(2+)](i) oscillations. Heparin inhibited NF-kappaB activation in Ang II-stimulated MCs that expressed either normal or chemically altered GAG. CONCLUSIONS: These findings suggest that alterations in HS-GAG chemistry or metabolism under pathological conditions, such as DN, may have direct functional consequences for the local effect of Ang II.     

Physiological and molecular characteristics of rat hypothalamic ventromedial nucleus glucosensing neurons

To evaluate potential mechanisms for neuronal glucosensing, fura-2 Ca(2+) imaging and single-cell RT-PCR were carried out in dissociated ventromedial hypothalamic nucleus (VMN) neurons. Glucose-excited (GE) neurons increased and glucose-inhibited (GI) neurons decreased intracellular Ca(2+) ([Ca(2+)](i)) oscillations as glucose increased from 0.5 to 2.5 mmol/l. The Kir6.2 subunit mRNA of the ATP-sensitive K(+) channel was expressed in 42% of GE and GI neurons, but only 15% of nonglucosensing (NG) neurons. Glucokinase (GK), the putative glucosensing gatekeeper, was expressed in 64% of GE, 43% of GI, but only 8% of NG neurons and the GK inhibitor alloxan altered [Ca(2+)](i) oscillations in approximately 75% of GK-expressing GE and GI neurons. Insulin receptor and GLUT4 mRNAs were coexpressed in 75% of GE, 60% of GI, and 40% of NG neurons, although there were no statistically significant intergroup differences. Hexokinase-I, GLUT3, and lactate dehydrogenase-A and -B were ubiquitous, whereas GLUT2, monocarboxylate transporters-1 and -2, and leptin receptor and GAD mRNAs were expressed less frequently and without apparent relationship to glucosensing capacity. Thus, although GK may mediate glucosensing in up to 60% of VMN neurons, other regulatory mechanisms are likely to control glucosensing in the remaining ones.     

The NMDA Type Glutamate Receptors Expressed by Primary Rat Osteoblasts Have the Same Electrophysiological Characteristics as Neuronal Receptors

Cells of mammalian bone express glutamate receptors. Functional N-methyl-D-aspartate (NMDA) receptors have been demonstrated in human, osteoblastic MG-63 cells, but currents in these cells, unlike those of mammalian neurons, are blocked by Mg(2+) in a voltage-insensitive manner. Differences between the characteristics of NMDA currents in bone cells and in neurons may reflect molecular variation of the receptors or associated molecules, with implications for the role(s) of glutamate in these different tissues and for targeting of ligands/antagonists. To determine whether NMDA receptors in primary bone cells are functional, and whether the currents carried by these receptors resemble those of MG-63 cells or those of mammalian neurons, we have applied the whole cell patch clamp technique to primary cultures of rat osteoblasts. In 0-Mg(2+) saline, 25% of cells showed a slowly developing inward current in response to bath perfusion with 1 mM or 100 microM NMDA. Antibodies against NMDA receptors stained approximately 26% of cells. When NMDA was applied by rapid superfusion, kinetics of the currents were similar to those of neuronal NMDA currents, reaching a peak within 20-30 ms. 1 mM Mg(2+) reduced current amplitude at negative holding potentials and caused the I-V relationship of the currents to adopt a 'J' shape rather than the linear relationship seen in the absence of added Mg(2+). Co-application of glycine (20 microM) with NMDA increased current amplitude by only 18%, suggesting that glycine is released from cells within the cultures. Currents were blocked by (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid. Fluorimetric monitoring of [Ca(2+)](i) using fura-2 showed that, in Mg(2+)-free medium, NMDA caused a sustained rise in [Ca(2+)](i) that could be reversed by subsequent application of MK-801. We conclude that rat femoral osteoblasts express functional NMDA receptors and that these receptors differ from those previously identified in MG-63 cells. NMDA receptors of primary osteoblasts show a 'classical' voltage-sensitive Mg(2+) block, similar to that seen in neuronal NMDA receptors, and will therefore function as detectors of coincident receptor activation and membrane depolarization.