[Survival and prognosis analysis of patients with Hodgkin lymphoma treated with standard treatment paradigm].

Objective: To analyze the survival and prognosis of Hodgkin lymphoma (HL) patients receiving standard first-line therapy. Methods: Data of clinical characteristics and treatment outcomes of patients with HL diagnosed in Cancer Hospital Chinese Academy of Medical Sciences (CHCAMS) from January 1st, 2000 to December 31st, 2018 who received standard first-line treatment were retrospectively analyzed and compared with that of HL patients who received treatment in the Surveillance, Epidemiology and End Results (SEER) database in the United States during the same period. Factors associated with freedom from progression (FFP) of patients in CHCAMS were analyzed. Treatment and survival data of patients with relapsed/refractory HL (r/rHL) who had failed the standard first-line treatment during the corresponding period in CHCAMS were collected to analyze the outcomes of salvage therapy. Results: A total of 764 HL patients in CHCAMS were included in this study. The median age was 30 years (range, 14-83 years), with 424 males and 340 females. By February 26th, 2022, the patients were followed-up for a median time of 111 months(range, 0.3-262.0 months). Lymphoma-specific survival (LSS) rate and overall survival (OS) rate at 10 years for HL patients in CHCAMS was 91.7% (95%CI: 89.5%-93.9%) and 87.1% (95%CI: 84.5%-89.8%), respectively. LSS and OS rate at 10 years for HL patients from SEER database was 86.8% (95%CI: 86.3%-87.2%) and 79.0% (95%CI: 78.5%-79.5%), respectively. The unadjusted LSS and OS rate for patients in CHCAMS were higher than those for patients from SEER database (both P<0.001). No significant difference was observed in LSS and OS rate (both P>0.05) between the two groups after adjustment. European Organization for Research and Treatment of Cancer staging system (early-stage unfavorable: HR=2.35, 95%CI: 1.13-4.89, P=0.023; advanced stage: HR=5.44, 95%CI: 2.62-11.30, P<0.001) and serum ß2 microglobulin (HR=1.67, 95%CI: 1.08-2.58, P=0.021) were influencing factors of FFP for patients in CHCAMS. The complete remission rate, median progression-free survival (PFS), 5-year PFS rate and 5-year OS rate for the 116 patients with r/rHL was 37.9% (95%CI: 29.6%-47.0%), 15.0 months (95%CI: 9.9-20.1 months), 29.9% (95%CI: 20.9%-38.9%) and 62.9% (95%CI: 54.1%-71.7%), respectively. Conclusions: The outcomes of HL patients receiving standard first-line treatment are excellent. However, the therapeutic effect of HL patients who incurrs disease progression or relapse after standard first-line treatment is not satisfying.

Effects of Ce, Sm and Yb on cavitation erosion of NAB alloy in 3.5% NaCl solution.

The influences of Ce, Sm and Yb on cavitation erosion of NAB alloy in 3.5% NaCl solution are evaluated using mass loss, SEM, 3D morphology and Tafel plot, respectively. The results show that the addition of Ce or Sm or Yb enhances the mechanical property of NAB alloy, and the sizes of κⅠ and κⅡ phases within NAB alloy decrease with adding Ce or Sm or Yb, resulting in the prevention of the propagation of the cracks caused by cavitation erosion initially originated at the phase boundaries between α and κ phases, and finally the cavitation erosion damage significantly decreases with adding Ce or Sm or Yb. The corrosion of NAB alloy in 3.5% NaCl solution can promote the cavitation erosion of NAB alloy, while the corrosion resistance of NAB alloy increases with the addition of Ce or Sm or Yb, and then the cavitation erosion resistance is accordingly improved with the addition of rare earth element.

[To take the initiative in the prevention and control of the coronavirus disease 2019 epidemic in the changing and unchanged strategies].

In early 2020, an outbreak of coronavirus disease 2019 (COVID-19) epidemic happened in China. In the following three months, 42 600 medical personnels and more than 9 000 public health employees were "rushed out" of their own position and onto Wuhan and other areas in Hubei Province. They helped to strengthen the treatment of severe cases and the isolation of mild cases, and actively carried out community screenings, and eventually won victory in the defense of Wuhan. During the normalization stage of prevention and control of the epidemic of COVID-19, China adopted the expanded preventive strategy by focusing on widely implement PCR testing, and integrate general and emergency departments to improve the performance of public health system. In this stage, China put out the cluster of epidemics that have occurred in several places one after another, and effectively controlled the spread of the epidemic in 2 to 3 incubation periods. In the stage of "dynamic zeroing", China emphasized the strategy of "grasping early, grasping the basics", focused on specific measures such as precise prevention in key areas. The rule of golden 24 hours was used to control the spread of the epidemic within one incubation period. During the epidemic, China continues to adopt active prevention and control strategies. This self-confidence and determination depends on adhering to the leadership of the Communist Party of China, the distinct essence of medical and health services, and significant advantages of social governance on health.

[Analysis of respiratory syncytial virus detection outcomes of 973 cases with severe respiratory infection during 2016-2019, Yuhang district of Hangzhou].

This study collected nasopharyngeal swab specimens from severe respiratory infection cases in First People's Hospital of Yuhang District during 2016-2019. Real-time PCR was used to detect respiratory syncytial virus (RSV). Rate of RSV positive detection were analysised in different age groups and different months. A total of 973 nasopharyngeal swab specimens of severe respiratory infection cases were collected, and the total positive rate of nucleic acid test of RSV was 6.47%; The detection rate of nucleic acid in male is higher than that in female, with no statistical differences (P=0.023). The positive rate of nucleic acid test was negatively correlated with age. The positive rate was 15.2% in the group aged 0-1 years and 12% in the group aged 1-2 years. There are obvious seasonal differences in the prevalence of RSV, human are easier to infect RSV in spring and winter.

[Intraobserver reproducibility of Ki-67 assessment of breast cancers based on digital slide].

Objective: To investigate the intra-observer reproducibility of Ki-67　　　assessment in breast cancers using three methods based on digital slide. Methods: Thirty cases of invasive breast cancer tissues were immunostained for Ki-67 by automatic stainer, and then scanned into digital pathological slides. Ki-67 positive index was measured individually by three pathologists using size-set visual assessment of hot spot (SSVAHS), size-set semi-automatic counting of hot spot(SSSACHS), and size-set automatic counting of hot spot (SSACHS), respectively, and repeated for 10 times. Intraclass correlation coefficient (ICC) of each assessment method was calculated, and the intraobserver reliability was classified as excellent, good, fair and poor according to ICC. Results: The ICC by 3 pathologists using SSVAHS was 0.832, 0.843 and 0.826, respectively, The ICC using SSSACHS was 0.926,0.938,0.929, and the ICC using SSACHS was 0.964, 0.971 and 0.968.The intraobserver reliability level of all three methods was excellent. Conclusion: The three methods of Ki-67 assessment achieve satisfactory intraobserver reproducibility, and the order of reproducibility from high to low is SSACHS, SSSACHS, and SSVAHS.

[Increased receptor activator of nuclear factor kappa B ligand expressed on B10 cells in rheumatoid arthritis].

OBJECTIVE: To detect receptor activator of nuclear factor kappa B ligand (RANKL) expressed on B10 cells in rheumatoid arthritis (RA) and to evaluate the correlation between RANKL-producing B10 cells in RA and clinical features and laboratory parameters, trying to reveal the possible role of B10 cells in the pathogenesis of RA and the potential mechanism of impaired immunosuppressive capacities. METHODS: 25 RA patients and 20 healthy volunteers were enrolled. These RA patients did not received treatment with glucocorticoids, disease-modifying anti-rheumatic drug and biologics during the recent half of a year. The levels of RANKL-producing B10 cells were measured by flow cytometry (FCM) and polymerase chain reaction (PCR). The correlation between the frequencies of RANKL-producing B10 cells in RA and clinical data, laboratory parameters were analyzed. The role of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) in inducing RANKL expression in B10 cells was evaluated by in vitro stimulation assay. Independent samples t test, Pearson and Spearman correlation were used for statistical analysis. RESULTS: B10 cells were capable of producing RANKL at a low level in health controls. The frequencies of RANKL-producing B10 cells were markedly higher in RA patients than in health controls (3.65%±1.59% vs. 2.25%±0.68%, P<0.01). The frequencies of these cells correlated positively with RA tender joint counts, swollen joint counts and disease activity score in 28 joints (DAS28) (r=0.479, P=0.035; r=0.519, P=0.008; r=0.526, P=0.019). However, no correlation was found between these cells and RA patient age, disease duration, or the levels of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF) and anti-citrullinated peptide antibody (ACPA). After in vitro stimulation by TNF-α, but not IL-1ß, B10 cells isolated from healthy donors demonstrated fundamentally upregulated expression of RANKL. CONCLUSION: Our studies showed the frequencies of RANKL-producing B10 cells were markedly higher in RA patients, and their frequencies were positively correlated with RA tender joint counts, swollen joint counts and DAS28. These findings suggested that B10 cells might be involved in RA bone destruction.

Genetic differentiation of Anisakis species (Nematoda: Anisakidae) in marine fish Priacanthus tayenus from Gulf of Thailand.

Members of the genus Anisakis are parasitic nematodes of the family Anisakidae. They are potential zoonotic parasites, causing anisakiasis in humans who consume raw or undercooked seafood (fish or squid) infected with the larvae of this nematode. In the present study, anisakid nematodes collected from the marine fish Priacanthus tayenus (Purplespotted big-eye) caught from the Gulf of Thailand were examined morphologically and characterized genetically by DNA sequence analysis. Sequence data from the mitochondrial cytochrome c oxidase subunit II (mtDNA cox2) gene were used to identify these nematodes to species level and to evaluate the phylogenetic relationship among various taxa. All the 15 third-stage larvae of Anisakis nematodes investigated in this study belonged to the same genetic lineage as the A. typica species complex (named here as A. typica sp. T - T for Thailand). Eight mtDNA cox2 haplotypes were revealed in the 15 isolates of this Anisakis from Thailand. The mtDNA cox2 haplotypes of A. typica sp. T from Thailand were genetically distinct from those of the A. typica sensu stricto. Taxonomic description of this A. typica sp. T as a distinct species however awaits the availability of adult specimens.

Angiostrongylus mackerrasae and A. cantonensis (Nematoda: Metastrongyloidea) belong to same genetic lineage: evidence from mitochondrial protein-coding genes.

Angiostrongylus mackerrasae is a parasitic nematode of rats found in Australia. When first reported, it was referred to as A. cantonensis. Recent molecular studies, including the mitochondrial genome, indicate that it is highly similar to A. cantonensis. These studies did not include A. malaysiensis, another member of the A. cantonensis species complex, for comparison. The present study examined the genetic distance and phylogenetic relationship between the component taxa (A. cantonensis, A. mackerrasae and A. malaysiensis) of the A. cantonensis species complex, based on the 12 protein-coding genes (PCGs) of their mitochondrial genome. Both the nucleotide and amino acid sequences were analysed. Angiostrongylus mackerrasae and A. cantonensis are members of the same genetic lineage and both are genetically distinct from A. malaysiensis. The genetic distance based on concatenated nucleotide sequences of 12 mt-PCGs between A. mackerrasae and A. cantonensis from Thailand is p = 1.73%, while that between the Thai and Chinese taxa of A. cantonensis is p = 3.52%; the genetic distance between A. mackerrasae and A. cantonensis from China is p = 3.70%. The results indicate that A. mackerrasae and A. cantonensis belong to the same genetic lineage, and that A. mackerrasae may be conspecific with A. cantonensis. It remains to be resolved whether A. mackerrasae is conspecific with A. cantonensis or undergoing incipient speciation.

Cytochrome c oxidase subunit I haplotype reveals high genetic diversity of Angiostrongylus malaysiensis (Nematoda: Angiostrongylidae).

The rat lungworm Angiostrongylus malaysiensis is a metastrongyloid nematode parasite. It has been reported in Malaysia, Thailand, Laos, Myanmar, Indonesia and Japan. In this study, A. malaysiensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed high COI haplotype diversity of A. malaysiensis from Thailand. The geographical isolates of A. malaysiensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. cantonensis. In the present study, five new haplotypes were identified in addition to the four haplotypes reported in the literature. Phylogenetic analysis revealed that four of these five new haplotypes - one from Mae Hong Song (northern region), two from Tak (western region) and one from Phang Nga (southern region) - formed a distinct clade with those from Phatthalung (southern region) and Malaysia. The haplotype from Malaysia was identical to that of Phatthalung (haplotype AM1). In general, the COI sequences did not differentiate unambiguously the various geographical isolates of A. malaysiensis. This study has confirmed the presence of high COI genetic diversity in various geographical isolates of A. malaysiensis. The COI gene sequence will be suitable for studying genetic diversity, population structure and phylogeography.

Genetic differentiation of Anisakis species (Nematoda: Anisakidae) in marine fish Priacanthus tayenus from Gulf of Thailand

@#Members of the genus Anisakis are parasitic nematodes of the family Anisakidae. They are potential zoonotic parasites, causing anisakiasis in humans who consume raw or undercooked seafood (fish or squid) infected with the larvae of this nematode. In the present study, anisakid nematodes collected from the marine fish Priacanthus tayenus (Purplespotted big-eye) caught from the Gulf of Thailand were examined morphologically and characterized genetically by DNA sequence analysis. Sequence data from the mitochondrial cytochrome c oxidase subunit II (mtDNA cox2) gene were used to identify these nematodes to species level and to evaluate the phylogenetic relationship among various taxa. All the 15 third-stage larvae of Anisakis nematodes investigated in this study belonged to the same genetic lineage as the A. typica species complex (named here as A. typica sp. T – T for Thailand). Eight mtDNA cox2 haplotypes were revealed in the 15 isolates of this Anisakis from Thailand. The mtDNA cox2 haplotypes of A. typica sp. T from Thailand were genetically distinct from those of the A. typica sensu stricto. Taxonomic description of this A. typica sp. T as a distinct species however awaits the availability of adult specimens.

Genetic diversity of infective larvae of Gnathostoma spinigerum (Nematoda: Gnathostomatidae) in freshwater swamp eels from Thailand.

Human gnathostomiasis is a food-borne zoonosis caused by a tissue nematode of the genus Gnathostoma. The disease is highly endemic in Asia, including Thailand. The freshwater swamp eel (Monopterus albus), the second intermediate host of the gnathostome nematode, has an important role in transmitting the infection in Thailand. Surveys on the infective larvae of Gnathostoma spinigerum based on morphological features in freshwater swamp eels have been performed continuously and reported in Thailand. However, there is still limited molecular data on intra-species variations of the parasite. In this study, a total of 19 third-stage larvae of morphologically identified G. spinigerum were collected from 437 liver samples of freshwater swamp eels purchased from a large wholesale market in Bangkok, Thailand. Molecular characterization based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was performed to elucidate their genetic variations and phylogenetic relationship. Among the 19 infective larvae recovered from these eels, 16 were sequenced successfully. Phylogenetic analyses inferred from the partial COI gene showed the presence of three distinct COI haplotypes. Our findings confirm the presence of G. spinigerum as the main species in Thailand.

Integrated analysis of gene expression profile and genetic variations associated with ovarian cancer.

OBJECTIVE: Ovarian cancer is the most lethal gynecologic cancer worldwide, since most patients are diagnosed at an advanced stage. To improve the early diagnosis and treatment of ovarian cancer, we performed a integrated analysis of transcription profile and genetic variations to study on the molecular pathogenesis in ovarian cancer. METHODS: mRNA expression profiles of ovarian cancer and normal controls downloaded from ArrayExpress database were applied to identify differentially expressed genes (DEGs). The chromosomal distributions of these DEGs were established using DAVID. Then, DNASeq data from the Cancer Genome Atlas (TCGA) were extracted to analyze gene mutational information including the number of mutations (mut), the number of mutational genes (mutG) and chromosomal distributions of mutations. Statistical method was offered to carrying on correlation analysis of gene mutations and differential expression. RESULTS: A total of 1732 DEGs were identified, and the chromosomal distributions of 97 genes were unknown. These DEGs were most significantly distributed on chromosome 4 with p value = 1.34E-7. Chromosome 1 enriched the most DEGs (11.56%). Statistical algorithm showed that DEGs presented significantly positive correlation with mut (p = 0.000009) and mutG (p = 0.00001). In 48.7% DEGs, gene mutations were found. CONCLUSIONS: We conducted scientific analysis on integration of DEGs in expression profiles and genetic mutations in ovarian cancer, displayed the correlation of differential expression and genetic variations. The result indicated that expression profiles were significantly correlated to genotype.

New hope for the treatment of osteoarthritis through selective inhibition of MMP-13.

Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients, and is characterized by progressive loss of articular cartilage that eventually leads to a complex process involving degradation of various components of the cartilage matrix, chief among them are the cartilage-specific type II collagen (CII) and aggrecan. While the loss of aggrecan is thought to be an early and reversible process, degradation of CII is considered to be irreversible and a key step in the loss of structural and functional integrity of cartilage. Among the various matrix metalloproteinases (MMPs), MMP-13 is specifically expressed in the cartilage of human OA patients and is not present in normal adult cartilage. It is the major collagenase in OA cartilage and has the highest activity against CII. However, the clinical utility of broad-spectrum MMP inhibitors developed for treatment of OA has been restricted by dose- and duration-dependent musculoskeletal side effects in humans. Consequently, selectively inhibiting the MMP-13 would seem to be an attractive therapeutic objective. This review mainly focuses on selective MMP-13 inhibitors development in terms of OA since the late 90s, in terms of synthetic compounds of low molecular mass incorporating specific zinc-binding groups, non-zinc-binding groups. In addition, dual inhibitors of MMP-13 and aggrecanase are also reviewed. Special emphasis is placed on logistic concerns for lead compound search as well as the structure-activity relationship (SAR) in this field. Through these methods, new hope is emerging for the treatment of OA through selective inhibition of MMP-13.

Phosphorylation promotes the desensitization of the opioid-induced Ca2+ increase in NG108-15 cells.

Using the fluorescent Ca2+ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca2+ concentration ([Ca2+]i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca2+]i increase, while exposure of desensitized cell to 50 mM K+ restores the response EK-induced, suggesting a role of intracellular Ca2+ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[N, O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4, 5-trisphosphate (IP3) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP3 generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca2+ influx by either verapamil or removal of extracellular Ca2+ or the depletion of Ca2+ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca2+ transporters involved in Ca2+ influx and Ca2+ release. Our results imply that emptying of intracellular Ca2+ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.

Inhibition of bradykinin-induced calcium increase by phosphatase inhibitors in neuroblastoma x glioma hybrid NG108-15 cells.

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.

Antagonistic effect of Na+ and Mg2+ on P2z purinoceptor-associated pores in dibutyryl cyclic AMP-differentiated NG108-15 cells.

The effect of replacement of extracellular Na+ with N-methyl-D-glucamine (NMG) on P2 receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15, cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 value of 230 microM. Replacement of Na+ with NMG as well as removal of Mg2+ from the bathing buffer potentiated ethidium bromide uptake, [Ca2+]i increase, and 45Ca2+ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg2+ to limit the amount of ATP4-, replacement of Na+ with NMG had no effect on the ATP-induced [Ca2+]i increase but caused a markedly larger [Ca2+]i increase when the calculated concentration of ATP4- was > 10 microM. The calculated EC50 value for ATP4- stimulation of the [Ca2+]i increase was 23 microM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca2+ release was the major pathway for the ATP-induced [Ca2+]i increase; both removal of Mg2+ and replacement of Na+ with NMG did not affect the action of ATP. These data suggest that ATP(4-)-promoted pores are antagonized by Na+ and Mg2+ in dibutyryl cyclic AMP-differentiated NG108-15 cells.

P2 purinoceptor-mediated inhibition of cyclic AMP accumulation in NG108-15 cells.

In neuroblastoma X glioma hybrid NG108-15 cells, P2 purinoceptor agonists inhibited forskolin-stimulated cyclic AMP accumulation with distinct selectivities and their activities could be partially reversed by P2 purinoceptor antagonists. The rank order of potency in inhibition of cyclic AMP accumulation was UTP > 2 methylthio-ATP (MeSATP) > benzoylbenzoic ATP (BzATP) = alpha, beta-methylene ATP (AMPCPP) > beta, gamma-methylene ATP (AMPPCP) > ATP > ADP > adenosine 5'-thiotriphosphate (ATP gamma S). Neither adenosine nor AMP caused any inhibitory effect on cyclic AMP accumulation. Pertussis toxin treatment of cells attenuated the inhibitory effect of UTP, MeSATP and ATP on cyclic AMP accumulation whereas it had no effect on the BzATP-induced response. In addition, P2-purinoceptor-mediated inhibition of cyclic AMP accumulation was insensitive to cytosolic Ca2+ concentration. The breakdown of cyclic AMP was enhanced by MeSATP but not by the addition of ATP, UTP and BzATP. Our results suggest that a pertussis toxin-sensitive Gi signalling pathway is directly coupled to the occupancy of P2u and P2y receptors in NG108-15 cells.

Heterologous desensitization of opioid-stimulated Ca2+ increase by bradykinin or ATP in NG108-15 cells.

Leucine-enkephalin (Leu-EK) dose-dependently elicited an increase in cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 of 1.2 microM via the phosphoinositide cascade in NG108-15 cells. Chronic treatment of cells with [D-Ala2,D-Leu5]enkephalin caused time-dependent homologous desensitization. In the presence of extracellular Ca2+, ATP as well as bradykinin stimulated significantly higher increases in inositol 1,4,5-trisphosphate (IP3) generation than did Leu-EK; however, the magnitude of intracellular Ca2+ pools increased after ATP stimulation, whereas bradykinin depleted intracellular pools. Hence, cells lost their [Ca2+]i response to Leu-EK if bradykinin was first added to induce a [Ca2+]i increase, whereas the response was unchanged if Leu-EK was added after addition of ATP. When Leu-EK was added simultaneously with bradykinin or ATP, an additive response was observed in IP3 generation; however, the rise in [Ca2+]i reached the same level as that induced by bradykinin or ATP alone. In the absence of extracellular Ca2+ in which the replenishment of intracellular pools was not possible, ATP displayed an inhibitory effect similar to that of bradykinin on the Leu-EK-induced [Ca2+]i increase. Prior treatment of cells with Leu-EK slightly heterologously desensitized the action of bradykinin, but had no effect on the ATP response. Our results suggest that a shared intracellular Ca2+ pool is sensitive to the opioid, bradykinin and P2-purinoceptor agonists; however, a defined pool of phosphatidylinositol 4,5-bisphosphate or a specific phospholipase C is responsible for each receptor.

Two distinct ATP signaling mechanisms in differentiated neuroblastoma x glioma hybrid NG108-15 cells.

The ATP signaling mechanism in neuroblastoma x glioma hybrid NG108-15 cells differentiated by exposure to dibutyryl-cAMP was characterized. In cells loaded with fura-2, ATP rapidly raised the cytosolic Ca2+ concentration ([Ca2+]i); the magnitude of the rise was inversely proportional to the extracellular Na+ concentration. Large increases in cytosolic Na+ concentration, measured with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate, were dose-dependently elicited by ATP. ATP also evoked the entry of ethidium bromide into cells, and this process was inhibited by Mg2+. Inositol-1,4,5-trisphosphate (IP3) generation induced by ATP was totally blocked by removal of extracellular Ca2+, but residual IP3 generation still remained in nondifferentiated cells. In addition, ATP produced a concentration-, time-, and Mg(2+)-dependent biphasic uptake of 45Ca2+. A range of nucleotides and ATP analogues, including CTP, UTP, and GTP, induced only 9-29% of the ATP response. However, adenosine 5'-thiotriphosphate evoked 79% of ATP-induced 45Ca2+ uptake. 45Ca2+ uptake elicited by ATP could be potently blocked by purinoceptor antagonists, but other tested reagents less effectively blocked the action of ATP. When bradykinin was used as an agonist, the [Ca2+]i rise was transient and was insensitive to the extracellular Na+ concentration. Na+ influx, entry of ethidium bromide, and 45Ca2+ uptake were unaffected by bradykinin. Furthermore, bradykinin-evoked IP3 generation was insensitive to extracellular Ca2+. Neither ATP nor bradykinin had any effect on cAMP levels within cells. These data suggest that ATP induces a [Ca2+]i rise in differentiated NG108-15 cells via two distinct Ca2+ influx mechanisms, i.e., a receptor-operated cation channel and pores formed by ATP4-. These mechanisms are distinct from those elicited by bradykinin.