Cholecystokinin receptors on guinea pig gastric chief cell membranes

Cholecystokinin (CCK) binding to its receptors on guinea pig gastric chief cell membranes was characterized with 125I-COOH terminal octapeptide of CCK (125I-CCK8). Specific binding of 125I-CCK8 to chief cell membranes was maximal at pH 6.0 and 30 degrees C after 180 min of incubation and reversible upon the addition of 10(-7) M unlabeled CCK8. CCK analogs such as CCK8, gastrin-I, and COOH-terminal tetrapeptide of CCK (CCK4) competitively inhibited the labeled CCK8 binding with the half maximal inhibitory concentration of 10(-10) M, 3 X 10(-7) M and 10(-6) M, respectively. Furthermore, guanine nucleotide analogs such as GTP gamma S and Gpp(NH)p also inhibited the labeled CCK8 binding to chief cell membranes. Scatchard analysis of the binding data at pH 6.0 revealed two orders of the binding sites and GTP gamma S decreased the binding by converting two binding sites of the receptors to only one site of lower affinity. These results suggest that there are specific receptors for CCK, which are coupled to a guanine nucleotide regulatory protein on guiea pig gastric chief cell membranes.     

Characterization of muscarinic acetylcholine receptors in the isolated gastric chief cells from guinea pig

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by assessing the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-induced pepsinogen secretion. CCh stimulated pepsinogen secretion in a dose dependent manner with the maximal and the half-maximal stimulatory concentrations at 10(-4) and 3 x 10(-6) M, respectively. Each of five different muscarinic receptor antagonists such as atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine reduced pepsinogen secretion stimulated by graded concentration of CCh, but did not alter the maximum secretion. The increase in concentration of each antagonist caused parallel rightward shift of the dose response curve to CCh. Schild analysis of the inhibition of CCh-induced pepsinogen secretion by the antagonists showed that pA2 values of atropine, scopolamine and 4-DAMP are 8.8, 9.2 and 9.0, respectively. On the other hand, pA2 values of pirenzepine and AF-DX116 are 6.5 and 5.9, respectively, suggesting that the muscarinic receptor mediating pepsinogen secretion from chief cells has a intermediate affinity for pirenzepine and a low affinity for AF-DX116. These results suggest that the muscarinic acetylcholine receptor mediating pepsinogen secretion from gastric chief cells is the M3 subtype.     

M3 muscarinic receptors mediate pepsinogen secretion via polyphosphoinositide hydrolysis in guinea pig gastric chief cells.

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.     

Leukotrienes stimulate pepsinogen secretion from guinea pig gastric chief cells by a nitric oxide-dependent pathway

Leukotrienes (LTs) are involved in many inflammatory conditions including gastric damage induced by nonsteroidal anti-inflammatory drugs. Although LTs stimulate acid secretion, the effect they exert on pepsinogen secretion is unknown. The aim of this study was to investigate whether LTs stimulate pepsinogen secretion by isolated chief cells and to identify the intracellular messengers that mediate this action. Isolated chief cells were incubated with concentrations of LTB₄, LTC₄, LTD₄, or LTE₄ ranging from 0.1 pmol/L to 10 μmol/L, and pepsinogen release, intracellular calcium and inositol(1,4,5)-trisphosphate (IP₃) concentrations were measured. Nitric oxide generation was determined by the amount of citrulline generated during incubation. All four LTs caused a concentration-dependent stimulation of pepsinogen secretion with 50% effective concentration of 0.05-0.1 nmol/L and a dose-dependent increase in cytoplasmic free calcium and IP₃ concentration. The LTB₄ and LTD₄ antagonists caused selective, concentration-dependent inhibition of LTB₄- and LTD₄-induced pepsinogen secretion, calcium mobilization, and IP₃ generation. All four LTs increased NO generation, and the effect was inhibited by LTB₄ and LTD₄ antagonists and an NO synthase inhibitor N^G-monomethyl-l-arginine and reversed by l-arginine. N^G-monomethyl-l-arginine caused a 50%–60% reduction of LT-induced pepsinogen release. Each of the four LTs caused a fivefold increase in 5′-cyclic guanosine monophosphate. LTs are powerful stimulators of pepsinogen secretion in isolated chief cells and act via occupancy of specific cell-surface receptors.     

Effects of ethanol on pepsinogen release from isolated guinea pig gastric chief cells

The effects of ethanol on pepsinogen release from isolated guinea pig gastric chief cells were investigated. Ethanol, at concentrations of 300 mM to 900 mM, dose-dependently stimulated increases in pepsinogen release, initial Ca influx rate and intracellular free Ca concentration ([Ca2+]i) without affecting chief cells' viability. The increases in all parameters were inhibited by La3+ or EGTA, but not by nifedipine or verapamil. COOH-terminal octapeptide of cholecystokinin (CCK8) also stimulated increases in pepsinogen release, initial Ca influx rate and [Ca2+]i. However, the increases were dependent on not only extracellular Ca2+ but also intracellular Ca2+ mobilization. These results suggest that ethanol stimulates Ca2+ influx via the mechanism different from that of CCK8 and thereby stimulates pepsinogen release from isolated guinea pig gastric chief cells.     

Expression of tumour necrosis factor receptors by bronchoalveolar cells in hypersensitivity pneumonitis.

Tumour necrosis factor receptors (TNFR) and the Fas receptor (FasR) have been implicated in the pathogenesis of interstitial lung diseases. The current authors examined the expression of TNFR-1, TNFR-2 and FasR by bronchoalveolar cells in hypersensitivity pneumonitis (HP). Cell surface receptor expression on bronchoalveolar lavage cells was analysed by immunocytochemistry in 11 HP patients, 11 idiopathic pulmonary fibrosis (IPF) patients and 10 controls. TNFR-1, TNFR-2 and FasR were expressed on a higher percentage of alveolar macrophages (AM) in HP compared with controls and IPF patients. TNFR-2 and FasR expression on lymphocytes was also higher in HP than in controls and in IPF. TNFR-1, TNFR-2 and FasR expression correlated positively with the percentage of lymphocytes, and negatively with the percentage of AM in HP. Expression of TNFR-1 on AM and TNFR-2 on lymphocytes correlated with the percentage of neutrophils in HP. In conclusion, this study shows evidence of altered expression of tumour necrosis factor superfamily receptors in hypersensitivity pneumonitis.     

Ethanol stimulates pepsinogen release by opening a Ca2+ channel of guinea pig gastric chief cells.

To better understand the mechanism by which ethanol causes gastric mucosal injury, the effects of ethanol on isolated guinea pig gastric chief cells were investigated in vitro. Ethanol at 300-900 mmol/L dose-dependently stimulated an increase in pepsinogen release without affecting chief cells' viability. Ethanol stimulated an increase in initial Ca2+ influx rate and intracellular Ca2+ concentration at the same concentrations as it stimulated pepsinogen release, whereas it did not stimulate cyclic adenosine monophosphate accumulation. Pepsinogen release stimulated by 900 mmol/L ethanol was almost equal to that stimulated by 10(-8) mol/L COOH terminal octapeptide of cholecystokinin. Ethanol did not stimulate an increase in the accumulation of inositol trisphosphates and tetrakisphosphates in [3H]inositol-labeled chief cells, whereas cholecystokinin octapeptide caused a significant increase in inositol trisphosphates and tetrakisphosphates. Ethanol-stimulated pepsinogen release and increases in the initial Ca2+ influx rate and intracellular Ca2+ concentration were inhibited by 0.5 mmol/L La3+ or 1 mmol/L ethylene glycol tetraacetic acid but were not inhibited by nifedipine or verapamil. These results suggest that the effect of ethanol on pepsinogen release from the gastric chief cells may be due to its opening of a Ca2+ channel that is sensitive to La3+ and that the pepsinogen releasing action of ethanol may be one of factors for ethanol-induced gastric mucosal injury.     

Soluble tumour necrosis factor receptors,tumour necrosis factor and interleukin-6 in serum in granulocytopenic patients with fever

Serum levels of TNF, IL-6 and soluble TNF receptors p55 and p75 (sTNFR-p55 and sTNFR-p75) were examined in 14 patients with acute myeloid leukaemia during 43 courses of chemotherapy. The patients experienced 30 episodes of fever which occurred during granulocytopenia (defined as granulocyte counts <0.2×10⁹/1) and six fever episodes when granulocyte counts were >1.0×10⁹/1. Febrile episodes were classified as microbiologically defined infection, clinically defined infection, and unexplained fever. Levels of bioactive IL-6 and immunoreactive TNF increased in response to fever during granulocytopenia, whereas bioactive TNF was not detected in any sample in this study. During granulocytopenia, both sTNFR rose significantly in microbiologically defined infection (P<0.01 for sTNFR-p55 and P<0.05 for sTNFR-p75), but not in the other two categories. The ratio of sTNFR-p55 to sTNFR-p75 was higher during febrile periods in granulocytopenia than in a non-granulocytopenic situation with granulocyte counts >1.0×10⁹/1 (P<0.01). We conclude that granulocytopenia affects release of the two sTNFR differently during febrile periods, and that release of sTNFR-p75 in response to fever is reduced during granulocytopenia, suggesting a role for the granulocytes in systemic release of sTNFR-p75.     

Circulating tumour necrosis factor alpha and soluble tumour necrosis factor receptors in patients with different patterns of rheumatoid synovitis

OBJECTIVE: To examine the relation between the serum levels of tumour necrosis factor alpha (TNFalpha), soluble tumour necrosis factor receptors (sTNF-R), and the histological pattern of rheumatoid synovitis. METHODS: An enzyme linked immunosorbent assay (ELISA) was used to measure TNFalpha, p55 sTNF-R, and p75 sTNF-R concentrations in the serum of 43 patients with rheumatoid arthritis (RA) and 34 patients with osteoarthritis (OA). RESULTS: Upon histological analysis two variants of rheumatoid synovitis emerged. Twenty six RA specimens presented only diffuse infiltrates of mononuclear cells. In the remaining 17 samples the formation of lymphocytic follicles with germinal centre-like structures was found. Serum concentrations of TNFalpha, p55 and p75 sTNF-R were raised in patients with RA compared with the OA control group (p<0.001 for all comparisons). Levels of TNFalpha, p55 and p75 sTNF-R were higher in the serum of patients with RA with follicular synovitis than in patients with diffuse synovitis (p<0.001, p<0.01, and p<0.05, respectively). Serum concentrations of TNFalpha, p55 and p75 sTNF-R correlated with markers of disease activity. CONCLUSION: Different histological types of rheumatoid synovitis associated with distinct serum levels of TNFalpha and sTNF-R reflect varying clinical activity of the disease and support the concept of RA heterogeneity.     

Difference in effects of sodium fluoride and cholecystokinin on pepsinogen secretion from isolated guinea pig gastric chief cells

In order to further investigate the precise mechanisms of cholecystokinin(CCK)-induced pepsinogen secretion from gastric chief cells, we compared the signal transducing mechanisms activated by CCK with those activated by sodium fluoride (NaF) in isolated guinea pig gastric chief cells. NaF stimulated a monophasic increase in diacylglycerol accumulation with a peak value observed at 15 sec, while CCK strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow and smooth increase in intracellular free Ca2+ concentration [( Ca2+]i) monitored by fura-2, while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of [Ca2+]i increase. Lanthanum chloride (La3+) effectively (unlike either nifedipine or verapamil) blocked NaF-stimulated increase in [Ca2+], but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. La3+ reduced NaF-or CCK-stimulated maximal pepsinogen secretion to 57.0 +/- 2.5% and 73.1 +/- 3.1% of control, respectively. These results suggest that NaF activates a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing an increase in diacylglycerol accumulation, Ca3+ influx and pepsinogen secretion in guinea pig gastric chief cells.     

Effects of CCK-receptor antagonists on CCK-stimulated pepsinogen secretion and calcium increase in isolated guinea pig gastric chief cells

The effects of CCK-receptor antagonists such as dibutyryl cyclic GMP (dbcGMP), L-364,718, and CR1409 on COOH-terminal octapeptide of CCK (CCK-8) -stimulated chief cell responses were examined using isolated guinea pig gastric chief cells. L-364,718 and CR1409 inhibited CCK-8-stimulated pepsinogen secretion over the same concentration range as they inhibited CCK-8-stimulated increases in intracellular Ca²⁺ concentration ([Ca²⁺]i), respectively. Schild analysis of the CCK dose-response curve indicates that L-364,718 and CR1409 exert their inhibitory effects on CCK-8-stimulated chief cell responses in a competitive manner. By contrast, dbcGMP inhibited not only CCK-8-but also carbachol-stimulated pepsinogen secretion. Furthermore, dbcGMP inhibited CCK-8-stimulated pepsinogen secretion more potently than the increases in [Ca²⁺]i. These results suggest that L-364,718 and CR1409 act as CCK-receptor antagonists, but dbcGMP has another inhibitory effect on pepsinogen secretion in addition to the effect as a CCK-receptor antagonist in guinea pig gastric chief cells.     

Human microvascular endothelial cells express receptors for platelet-derived growth factor.

Endothelial cells have been widely thought to be unresponsive to platelet-derived growth factor (PDGF, a major growth factor released from stimulated platelets at the sites of vascular insults) and devoid of PDGF receptors. Nevertheless, in examining the growth-factor responses of microvascular endothelial cells isolated from human omental adipose tissue, we were surprised to detect PDGF-induced tyrosine phosphorylation of a 180-kDa glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of 125I-labeled PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors per cell with a Kd of 0.14 nM. PDGF stimulated tyrosine phosphorylation of PDGF receptors and other cellular proteins in a dose- and time-dependent manner, with half-maximal receptor phosphorylation occurring at 0.3 nM recombinant human PDGF (B chain) and a less than or equal to 1-min exposure to PDGF. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kDa protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. Microvascular endothelial cells play a central role in neovascularization required for wound healing and solid tumor growth. Thus, the discovery of functional PDGF receptors on human microvascular endothelial cells suggests a direct role for PDGF in this process.     

Interleukin-1beta and tumour necrosis factor-alpha increase microvascular leakage in the guinea pig trachea

OBJECTIVE: Airway microvascular leakage is considered to be an important component of airway inflammation in asthma. In the present study we examined the effect of interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNFalpha) on airway microvascular leakage in vivo. METHODOLOGY: Tracheal Evans blue extravasation was examined in an isolated tracheal segment, in anaesthetized mechanically ventilated guinea pigs. Baseline tracheal microvascular leakage was measured in five animals. As a control group for aerosol challenge, the isolated tracheal segment (n = 5) underwent saline aerosol challenge. To test whether a combination of IL-1beta (10 ng/mL) and TNFalpha (100 ng/mL) induced Evans blue extravasation, the trachea was exposed to an aerosol of these cytokines (n = 5). As a positive control the tracheal segment was challenged with histamine aerosol (5 x 10(-2) mol) (n = 3). All aerosol challenges were for 1 min. RESULTS: TNFalpha and IL-1beta aerosol challenge significantly increased Evans blue extravasation (28.9 +/- 1.6 microg/g wet tissue, mean +/- SE) compared to saline challenge (13.8 +/- 3.0 microg/g; P < 0.05). Tracheal dye extravasation without aerosol challenge, was not significantly different from saline-challenged animals (17.5 +/- 2.9 and 13.8 +/- 3.0 microg/g, respectively). Histamine significantly increased Evans blue extravasation (50.1 +/- 4.8 microg/g; P < 0.05) compared to saline challenge. CONCLUSION: Pro-inflammatory cytokines, TNFalpha and IL-1beta are able to induce significant microvascular leakage in the guinea pig trachea.     

Role of myosin light-chain kinase and protein kinase C in pepsinogen secretion from guinea pig gastric chief cells in monolayer culture

We evaluated the role of myosin light-chain kinase (MLCK) and protein kinase C (PKC) in pepsinogen secretion from guinea pig gastric chief cells using a monolayer culture system of chief cells and an enzyme immunoassay system for guinea pig pepsinogen. An MLCK inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), significantly inhibited both the basal pepsinogen secretion and the secretion by carbamylcholine chloride (carbachol) or ionomycin without affecting intracellular free Ca²⁺ concentration ([Ca²⁺]i), but not by 12-O-tetradecanoylphorbol-13-acetate (TPA) or forskolin. A PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), significantly reduced the pepsinogen secretion by carbachol or TPA, but not by forskolin or ionomycin, and did not affect the basal secretion and the [Ca²⁺]i elevated by carbachol or ionomycin. We concluded that: (1) MLCK plays an important role in basal and drug-stimulated pepsinogen secretion, (2) MLCK is involved in the Ca²⁺-dependent intracellular pathway but not in the cyclic adenosine monophosphate (cAMP) dependent pathway, (3) PKC is irrelevant to activation of MLCK, and (4) increases in cAMP and [Ca²⁺]i are independent of activation of PKC.This study was supported by Grant (03670371) from the Ministry of Education, Japan.     

Desensitization of platelet-activating factor receptors, induced by inflammation in guinea pig ileal smooth muscle cells

Platelet-activating factor (PAF) is involved in the pathophysiology of motility changes induced by intestinal inflammation. The aim of this study was to evaluate a possible desensitization of PAF receptors in guinea pig intestinal smooth muscle cells after experimental inflammation induced by trinitrobenzenesulfonic acid (TNB). Saline or TNB (80 mg/kg) was injected in the intestinal lumen, and the animals were killed 6 days later. Smooth muscle cells from the circular layer were isolated, and cell contraction induced by PAF was measured. In cells from saline-treated animals, PAF induced a maximal cell contraction at 10 nmol/L and half-maximal contraction (EC₅₀) was 9 ± 0.2 pmol/L. After TNB injection, the maximal contraction induced by PAF was observed at 1000 nmol/L and EC₅₀ was 300 ± 70 pmol/L, indicating a 2 logmol/L right shift of the concentration-response curve of PAF. When animals were treated with the PAF antagonist, 20 mg/kg BN52021, or with 2 mg/kg indomethacin for the 6 days after TNB instillation, the right-sided shift of the concentration-response curve of PAF did not occur. Desensitization of PAF receptors occurring in intestinal smooth muscle cells after TNB instillation could be mediated in vivo by PAF itself via a prostaglandin-dependent pathway.     

Sharing of biological effect and receptors between guinea pig insulin and platelet-derived growth factor.

Insulins from the hystricomorphs (guinea pig, porcupine, coypu, and casiragua) at high concentration stimulate DNA synthesis in human fibroblasts to a greater level than other mammalian insulins or insulin-like growth factors (IGFs). 125I-Labeled guinea pig insulin binds to a specific receptor and this binding is competed for by hystricomorph insulins but not by porcine insulin or IGFs. Fetal bovine serum also inhibits the binding of 125I-labeled guinea pig insulin and is more potent than fetal bovine plasma, in concordance with their relative potencies for growth stimulation in human fibroblasts. Of several other known growth factors tested, only platelet-derived growth factor (PDGF) inhibits binding of 125I-labeled guinea pig insulin. Four preparations of PDGF that vary in purity and potency for the stimulation of DNA synthesis in human fibroblasts over a 1,000-fold range compete with binding of 125I-labeled guinea pig insulin in proportion to their biological potencies. The purest preparation of PDGF is able to inhibit binding of 125I-labeled guinea pig insulin by 50% at 15 ng/ml (0.25 nM). Biologically, guinea pig insulin, like PDGF, exhibits a synergistic effect with plasma in initiating DNA synthesis in human fibroblasts; this effect is not observed with other mammalian insulins or IGFs. Thus, hystricomorph insulins appear to be mediating their growth-promoting effect through a different receptor and mechanism than other mammalian insulins or IGFs. Further, hystricomorph insulins may be sharing the mechanism of action for their growth effects with PDGF, perhaps suggesting some relationship between these peptides from very different sources.