Kinase Mutations and Imatinib Response in Patients With Metastatic Gastrointestinal Stromal Tumor.

PURPOSE: Most gastrointestinal stromal tumors (GISTs) express constitutively activated mutant isoforms of KIT or kinase platelet-derived growth factor receptor alpha (PDGFRA) that are potential therapeutic targets for imatinib mesylate. The relationship between mutations in these kinases and clinical response to imatinib was examined in a group of patients with advanced GIST. PATIENTS AND METHODS: GISTs from 127 patients enrolled onto a phase II clinical study of imatinib were examined for mutations of KIT or PDGFRA. Mutation types were correlated with clinical outcome. RESULTS: Activating mutations of KIT or PDGFRA were found in 112 (88.2%) and six (4.7%) GISTs, respectively. Most KIT mutations involved exon 9 (n = 23) or exon 11 (n = 85). All KIT mutant isoforms, but only a subset of PDGFRA mutant isoforms, were sensitive to imatinib, in vitro. In patients with GISTs harboring exon 11 KIT mutations, the partial response rate (PR) was 83.5%, whereas patients with tumors containing an exon 9 KIT mutation or no detectable mutation of KIT or PDGFRA had PR rates of 47.8% (P = .0006) and 0.0% (P < .0001), respectively. Patients whose tumors contained exon 11 KIT mutations had a longer event-free and overall survival than those whose tumors expressed either exon 9 KIT mutations or had no detectable kinase mutation. CONCLUSION: Activating mutations of KIT or PDGFRA are found in the vast majority of GISTs, and the mutational status of these oncoproteins is predictive of clinical response to imatinib. PDGFRA mutations can explain response and sensitivity to imatinib in some GISTs lacking KIT mutations.

The impacts of a global pandemic on the efficacy and stability of contemporary wildlife conservation: South Africa as a case study.

Conservationists speculated on potential benefits to wildlife of lockdown restrictions because of the COVID-19 pandemic but voiced concern that restrictions impeded nature conservation. We assessed the effects of lockdown restrictions on biodiversity conservation in South Africa, a biodiverse country with economic inequality and reliance on wildlife resources. We solicited expert opinion using the IUCN's Threats Classification Scheme to structure a questionnaire and illustrated responses with individual case studies from government parastatal and non-governmental conservation organisations. The most highly reported threats were biological resource use, residential/commercial developments, invasive species, and human intrusions. The trends reported by 90 survey respondents were supported by case studies using environmental compliance data from parastatal conservation organisations. Lack of tourism revenue and funding were cited as hindrances to conservation. Mechanisms to prevent environmental degradation in the face of global emergencies must be implemented and 'ring-fenced' to ensure conservation is not a casualty during future global crises.

Quality of life following laparoscopic Nissen fundoplication: assessing short-term and long-term outcomes.

AIM: To investigate the quality of life following laparoscopic Nissen fundoplication by assessing short-term and long-term outcomes. METHODS: From 1992 to 2005, 249 patients underwent laparoscopic Nissen fundoplication. Short-term outcome data including symptom response, side effects of surgery, endoscopy, and patient's perception of overall success were collected prospectively. Long-term outcomes were investigated retrospectively in patients with a median follow-up of 10 years by assessment of reflux symptoms, side effects of surgery, durability of antireflux surgery, need for additional treatment, patient's perception of success, and quality of life. Antireflux surgery was considered a failure based on the following criteria: moderate to severe heartburn or regurgitation; moderate to severe dysphagia reported in combination with heartburn or regurgitation; regular proton pump inhibitor medication use; endoscopic evidence of erosive esophagitis Savary-Miller grade 1-4; pathological 24-h pH monitoring; or necessity to undergo an additional surgery. The main outcome measures were short- and long-term cure rates and quality of life, with patient satisfaction as a secondary outcome measure. RESULTS: Conversion from laparoscopy to open surgery was necessary in 2.4% of patients. Mortality was zero and the 30-d morbidity was 7.6% (95%CI: 4.7%-11.7%). The median postoperative hospital stay was 2 d [interquartile range (IQR) 2-3 d]. Two hundred and forty-seven patients were interviewed for short-term analysis following endoscopy. Gastroesophageal reflux disease was cured in 98.4% (95%CI: 95.9%-99.6%) of patients three months after surgery. New-onset dysphagia was encountered postoperatively in 13 patients (6.7%); 95% reported that the outcome was better after antireflux surgery than with preoperative medical treatment. One hundred and thirty-nine patients with a median follow-up of 10.2 years (IQR 7.2-11.6 years) were available for a long-term evaluation. Cumulative long-term cure rates were 87.7% (81.0%-92.2%) at 5 years and 72.9% (64.0%-79.9%) at 10 years. Gastrointestinal symptom rating scores and RAND-36 quality of life scores of patients with treatment success were similar to those of the general population but significantly lower in those with failed antireflux surgery. Of the patients available for long-term follow-up, 83% rated their operation a success. CONCLUSION: For the long-term, our results indicate decreasing effectiveness of laparoscopic antireflux surgery, although most of the patients seem to have an overall quality of life similar to that of the general population.

Inverse Laplace Transform of Multidimensional Relaxation Data Without Non-Negativity Constraint.

An algorithm based on Tikhonov regularization in generalized form is described to perform an inverse Laplace transform of multidimensional data without a non-negativity (NN) constraint for spectrum conditioning. Uniform penalty (UP) regularization is used to reduce the requirement for NN, and a further penalty is introduced for zero-crossing (ZC) of the spectrum. This ZC term is weighted with the slope of the curve, which does not prevent negative modes in the spectrum but makes nonphysical undershooting in the vicinity of narrow peaks more expensive. The performance of this algorithm is demonstrated using synthetic data, and the optimization of the free parameters for calculating the regularization matrix is discussed.

MMP-1 expression has an independent prognostic value in breast cancer.

BACKGROUND: Breast cancer consists of a variety of tumours, which differ by their morphological features, molecular characteristics and outcome. Well-known prognostic factors, e.g. tumour grade and size, Ki-67, hormone receptor status, HER2 expression, lymph node status and patient age have been traditionally related to prognosis. Although the conventional prognostic markers are reliable in general, better markers to predict the outcome of an individual tumour are needed. Matrix metalloproteinase-1 (MMP-1) expression has been reported to inversely correlate with survival in advanced cancers. In breast cancer MMP-1 is often upregulated, especially in basal-type breast tumours. The purpose of this retrospective study was to analyse MMP-1 expression in breast cancer cells and in cancer associated stromal cells and to correlate the results with traditional prognostic factors including p53 and bcl-2, as well as to patient survival in breast cancer subtypes. METHODS: Immunohistochemical analysis of MMP-1, ER, PR, Ki-67, HER2, bcl-2, p53 and CK5/6 expression was performed on 125 breast cancers. Statistical analyses were carried out using Kruskal-Wallis and Mann-Whitney -tests. In pairwise comparison Bonferroni-adjustment was applied. Correlations were calculated using Spearman rank-order correlation coefficients. Kaplan-Meier survival analyses were carried out to compare breast cancer-specific survival curves. Factors significantly associated with disease-specific survival in univariate models were included in multivariate stepwise. RESULTS: Positive correlations were found between tumour grade and MMP-1 expression in tumour cells and in stromal cells. P53 positivity significantly correlated with MMP-1 expression in tumour cells, whereas HER2 expression correlated with MMP-1 both in tumour cells and stromal cells. MMP-1 expression in stromal cells showed a significant association with luminal A and luminal B, HER2 overexpressing and triple-negative breast cancer subtypes. CONCLUSIONS: The most important finding of this study was the independent prognostic value of MMP-1 as well as Ki-67 and bcl-2 expression in tumour cells. Our study showed also that both tumoural and stromal MMP-1 expression is associated with breast tumour progression and poor prognosis. A significant difference of MMP-1 expression by cancer associated stromal cells in luminal A, luminal B and triple-negative breast cancer classes was also demonstrated.

Metabotropic glutamate receptor mGlu2 is resistant to homologous agonist-induced desensitization but undergoes protein kinase C-mediated heterologous desensitization.

To investigate the susceptibility of the group II metabotropic glutamate receptor mGlu2 to agonist-induced desensitization, the receptor was stably expressed in Chinese hamster ovary (CHO-mGlu2) or C6 glioma cells (C6-mGlu2). Exposure of CHO-mGlu2 cells to the group II mGlu receptor agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG-1; 10 µM) for up to 15 h did not affect the subsequent ability of LCCG-1 to inhibit forskolin-stimulated cAMP accumulation. Similarly, in C6-mGlu2 cells, prolonged exposure to LCCG-1 also did not affect the subsequent ability of LCCG-1 to inhibit cAMP formation. In contrast, exposure of CHO-mGlu2 cells to the protein kinase C activator phorbol myristate acetate (PMA) suppressed the ability of LCCG-1 to inhibit cAMP formation. Using an in vitro model of group II mGlu receptor activity, the hemisected neonatal rat spinal cord preparation, the ability of the selective group II agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC) to depress the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) did not desensitize when applied for up to 2 h. Together these results indicate that in contrast to most G protein-coupled receptors, the mGlu2 receptor is resistant to agonist-induced homologous desensitization, and that in vitro data suggests that resistance to desensitization is a physiologically relevant property of this mGlu receptor subtype.

Long-term results from a randomized phase II trial of standard- versus higher-dose imatinib mesylate for patients with unresectable or metastatic gastrointestinal stromal tumors expressing KIT.

PURPOSE: The outcome of patients diagnosed with advanced gastrointestinal stromal tumor (GIST) and treated long-term with imatinib mesylate is unknown. A previous report of a randomized phase II trial of imatinib mesylate in patients with incurable GIST detailed high response rates at both the 400 and the 600 mg/d dose levels. We conducted a long-term analysis of patients treated on the trial, including patients followed during an extension phase, to evaluate survival, patterns of failure, and potential prognostic factors, including tumor mutational status. PATIENTS AND METHODS: Patients with advanced GIST were enrolled onto an open-label, multicenter trial and were randomly assigned (1:1) to receive imatinib 400 versus 600 mg/d. Data were prospectively collected on KIT mutational status, total tumor area, and other potential prognostic factors. Patients were followed for a median of 63 months. RESULTS: One hundred forty-seven patients were enrolled: 73 were in arm A (imatinib 400 mg/d), and 74 were in arm B (imatinib 600 mg/d). Response rates, median progression-free survival, and median overall survival were essentially identical on both arms, and median survival was 57 months for all patients. Forty-one patients overall (28%) remained on the drug long-term. Female sex, the presence of an exon 11 mutation, and normal albumin and neutrophil levels were independently associated with better survival. CONCLUSION: Nearly 50% of patients with advanced GIST who were treated with imatinib mesylate survived for more than 5 years, regardless of a 400 or 600 mg/d starting dose.

Prognostic significance of matrix metalloproteinases-1, -2, -7 and -13 and tissue inhibitors of metalloproteinases-1, -2, -3 and -4 in colorectal cancer.

Strong expression of many matrix metalloproteinases (MMPs) has been related to poor survival of colorectal cancer (CRC) patients. The expression of tissue inhibitors of metalloproteinases (TIMPs) has been associated with both a beneficial and a poor outcome and there is thus a need to further clarify the significance of MMPs and TIMPs in CRC. The prognostic significance of 4 MMPs and TIMPs in CRC was evaluated. Formalin-fixed, paraffin-embedded tissue arrayed samples of 351 patients with primary colon or rectal cancer of Dukes' stages A-D were selected for immunohistochemical staining of MMP-1, -2, -7 and -13, and TIMP-1, -2, -3 and -4. High expression of MMP-2 in the malignant epithelium as well as in the surrounding stroma was associated with reduced survival of colon cancer patients. Strong epithelial and stromal cytoplasmic staining of TIMP-3 was associated with a longer survival in rectal cancer patients, and here the interobserver variation for evaluating the degree of staining was lower than for epithelial staining. Strong stromal cytoplasmic staining of TIMP-4 predicted longer survival of rectal cancer patients. Multivariate analysis showed that stromal cytoplasmic TIMP-3 staining was the only marker of independent prognostic value. MMP-2 might be a useful prognostic marker in colon cancer, and TIMP-3 and TIMP-4 in rectal cancer, but the findings associated with stromal staining should be interpreted with some caution. Different biologic behavior or different genetic development may explain the differences between colon and rectal cancers regarding the expression of MMP-2, TIMP-3 and TIMP-4.

Mechanisms of glutamate receptor induced proliferation of astrocytes.

Astrocytes express mainly metabotropic glutamate receptor 3 and metabotropic glutamate receptor 5 receptor subtypes, which show opposing effects on cellular proliferation upon activation. In this study, we investigated the mechanisms by which activation of these receptors modulates astrocyte proliferation. Activation of metabotropic glutamate receptor 5 with (S)-3,5-dihydroxyphenylglycine increased phospholipase D activity in astrocytes as well as astrocyte proliferation. The 3,5-dihydroxyphenylglycine-induced proliferation was inhibited in the presence of the metabotropic glutamate receptor 5 antagonist (2-methyl-6-(phenylethynyl)pyridine), the protein kinase C inhibitor GF109203X, brefeldin A and 1-butanol. Activation of metabotropic glutamate receptor 3 with (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine-IV (DCG-IV) inhibited astrocyte proliferation without affecting metabotropic glutamate receptor 5-mediated phospholipase D activity. Metabotropic glutamate receptor 3 activation, however, only partially inhibited metabotropic glutamate receptor 5-mediated proliferation. In conclusion, metabotropic glutamate receptor 5 stimulates astrocyte proliferation via a protein kinase C-phospholipase D-phosphatidic acid-dependent pathway, whereas metabotropic glutamate receptor 3-mediated inhibition of astrocyte proliferation does not involve phospholipase D, and is independent of metabotropic glutamate receptor 5-mediated effects.

Molecular correlates of imatinib resistance in gastrointestinal stromal tumors.

PURPOSE: Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT or platelet-derived growth factor alpha (PDGFRA) kinases, which are targets for imatinib. In clinical studies, 75% to 90% of patients with advanced GISTs experience clinical benefit from imatinib. However, imatinib resistance is an increasing clinical problem. PATIENTS AND METHODS: One hundred forty-seven patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib. Specimens from pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. Secondary kinase mutations of KIT or PDGFRA that were identified in imatinib-resistant GISTs were biochemically profiled for imatinib sensitivity. RESULTS: Molecular studies were performed using specimens from 10 patients with primary and 33 patients with secondary resistance. Imatinib-resistant tumors had levels of activated KIT that were similar to or greater than those typically found in untreated GISTs. Secondary kinase mutations were rare in GISTs with primary resistance but frequently found in GISTs with secondary resistance (10% v 67%; P = .002). Evidence for clonal evolution and/or polyclonal secondary kinase mutations was seen in three (18.8%) of 16 patients. Secondary kinase mutations were nonrandomly distributed and were associated with decreased imatinib sensitivity compared with typical KIT exon 11 mutations. Using RNAi technology, we demonstrated that imatinib-resistant GIST cells remain dependent on KIT kinase activity for activation of critical downstream signaling pathways. CONCLUSION: Different molecular mechanisms are responsible for primary and secondary imatinib resistance in GISTs. These findings have implications for future approaches to the growing problem of imatinib resistance in patients with advanced GISTs.

Synthesis and pharmacology of willardiine derivatives acting as antagonists of kainate receptors.

The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10) is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and AMPA receptors analogues of willardiine with substituents at the N3 position of the uracil ring were synthesized. The N3-4-carboxybenzyl substituted analogue (38c) was found to be equipotent at AMPA and GLUK5-containing kainate receptors in the neonatal rat spinal cord. The N3-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLUK5 subunit containing kainate receptor antagonist when tested on native rat and human recombinant AMPA and kainate receptor subtypes. The GLUK5 kainate receptor antagonist activity was found to reside in the S enantiomer (44a) whereas the R enantiomer (44b) was almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have enhanced potency and selectivity for GLUK5.

Analysis of mGluR1a constitutive internalization using a pulse-chase enzyme-linked immuno-sorbant assay (ELISA).

The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse-chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse-chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse-chase labelling procedure. The application of the pulse-chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse-chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.

Increased proliferation activity measured by immunoreactive Ki67 is associated with survival improvement in rectal/recto sigmoid cancer.

AIM: To assess the expression of Ki67 as prognosticator in rectal/recto sigmoid cancer. METHODS: Samples from 146 patients with rectal and recto sigmoid cancer were studied for expression of Ki67 and its prognostic significance in comparison with clinico-pathological predictors of survival. Formalin-fixed, paraffin-embedded tissues from 6 (4.1%) patients with T1, 26 (17.8%) with T2, 94 (64.4%) with T3, and 20 (13.7%) with T4 tumors were studied. Ki67 expression was determined immunohistochemically. Samples were divided according to mean value into high (>40%) and low (< or =40%) expression. Areas of extensive proliferation (>50%) were defined as "hot spot" areas. RESULTS: Hot spot areas were present in samples regardless of histopathological grade. Lower TNM and Dukes stage and higher expression of Ki67 and presence of Ki67 hot spot areas in histopathological samples were associated with better survival, whereas no association was observed with histopathological grade (P = 0.78). In Cox multivariate regression analysis, significant prognostic factors were Dukes stage (P<0.001), presence of lymph node metastases (P = 0.015), age (P = 0.035) and presence of Ki67 hot spot areas (P = 0.044). CONCLUSION: Proliferative activity as measured by Ki67 in rectal cancer is associated with survival improvement compared with patients with low Ki67. Areas of prognostically significant increased proliferation were found independently of histopathological tumor grade.

The significance of tumor markers for proliferation and apoptosis in predicting survival in colorectal cancer.

PURPOSE: Clinicopathologic staging is even today the best prognostic factor in both colon and rectal cancers. There is still considerable variation in survival within the stages. To find other prognostic indicators we investigated six biologic markers associated with apoptosis and cell proliferation. METHODS: Formalin-fixed, paraffin-embedded tissue samples of 363 patients with primary colon or rectal cancer of Dukes Stages A to D were chosen for immunohistochemical staining of five tumor markers: bcl-2, p53, Ki-67, cyclin D1, and carcinoembryonic antigen. Also, the number of apoptotic cells was studied by the terminal deoxynucleotidyl transferase-mediated D: -UTP nick end labeling method in 347 cases. The study was done on specially prepared tissue arrays. RESULTS: In rectal cancer, patients with a Ki-67 labeling index of 5 percent or higher had a better prognosis than those with a lower index. Also, positive cytoplasmic p53 expression predicted a favorable outcome in rectal cancer. In colon cancer, positive nuclear staining of cyclin D1 reflected better survival. Weak and moderate staining of carcinoembryonic antigen correlated with better prognosis than strong staining, but negative staining predicted poor outcome. High apoptotic index of 100 or higher correlated with poor prognosis in colon cancer. However, in rectal cancer, the trend was the opposite. Bcl-2 staining tended to be more intense in samples of patients living 5 years or longer compared with those with worse prognosis. CONCLUSIONS: Colon cancer and rectal cancer seem to have different biologic behavior, at least with respect to apoptosis, cytoplasmic p53 expression, and perhaps Ki-67 and carcinoembryonic antigen. Further studies are needed to clarify the significance of these factors.

The influence of training level and surgical experience on survival in colorectal cancer.

BACKGROUND AND AIMS: The effect of surgical training level, experience, and operation volume on complications and survival in colorectal cancer during a 10-year period in a medium-volume university hospital was retrospectively studied. PATIENTS AND METHODS: Four hundred and fifty-six patients were resected for primary colorectal adenocarcinoma during the 10-year period of 1981-1990, and of these, 387 patients underwent resection with curative intent. The surgeons were divided into three groups according to training level and volume: group 1, surgeons in training and other surgeons operating annually on only 1-4 patients; group 2, surgeons specializing in gastrointestinal surgery (average annual volume 4-13 operations); group 3, specialists in gastrointestinal surgery (average annual volume 3-8 operations). Postoperative morbidity and mortality rates, as well as long-term survival rates, were analysed, and comparisons were made between the patients in the three groups. RESULTS: There were no statistically significant differences between the three groups in postoperative morbidity or mortality. Cancer-specific 5-year survival rate of all patients was 57%, and that of those resected in the aforementioned three groups was 51%, 63%, and 55%, respectively, P=0.087. The 5-year survival rates for colon cancer were 59% (total), 52%, 69%, and 58%, respectively, P=0.067, and for rectal cancer were 51% (total), 42%, 53%, and 52%, respectively, P=0.585. CONCLUSION: There were no significant differences in the rates of postoperative mortality, morbidity, and long-term overall survival between the volume groups. However, in patients with colon cancer, there was a trend for better survival for those operated on by the surgeons specializing in gastrointestinal surgery, and in rectal cancer patients, a tendency of fewer local recurrences in those operated on by the specialist surgeons.

Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous Gq/11-coupled receptors.

In this study we characterized the heterologous desensitization and internalization of the metabotropic glutamate receptor 1 (mGluR1) splice variants mGluR1a and mGluR1b following activation of endogenous G(q/11)-coupled receptors in HEK293 cells. Agonist activation of M1 muscarinic acetylcholine or P2Y1 purinergic receptors triggered the PKC- and CaMKII-dependent internalization of mGluR1a. In co-immunoprecipitation studies, both glutamate and carbachol increased the association of GRK2 with mGluR1a. Co-addition of the protein kinase C (PKC) inhibitor GF109203X and the Ca(2+) calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 blocked the ability of glutamate and carbachol to increase the association of GRK2 with mGluR1a. Glutamate also increased the association of GRK2 with mGluR1b, whereas carbachol did not. However, unlike mGluR1a, glutamate-stimulated association of GRK2 with mGluR1b was not reduced by PKC/CaMKII inhibition. Pretreatment of cells expressing mGluR1a or mGluR1b with carbachol rapidly desensitized subsequent glutamate-stimulated inositol phosphate accumulation. The carbachol-induced heterologous desensitization and internalization of mGluR1a was blocked by LY367385, an mGluR1a antagonist with inverse agonist activity. Furthermore, LY367385 blocked the ability of carbachol to increase the association of GRK2 with mGluR1a. On the other hand, LY367385 had no effect on the carbachol-induced desensitization and internalization of the nonconstitutively active mGluR1b splice variant. These results demonstrate that the internalization of mGluR1a, triggered homologously by glutamate or heterologously by carbachol, is PKC/CaMKII-, GRK2-, arrestin-, and clathrin-dependent and that PKC/CaMKII activation appears to be necessary for GRK2 to associate with mGluR1a. Furthermore, the heterologous desensitization of mGluR1a is dependent upon the splice variant being in an active conformation.

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists.

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins.

Novel metabotropic glutamate receptor negatively coupled to adenylyl cyclase in cultured rat cerebellar astrocytes.

Several excitatory amino acid ligands were found potently to inhibit forskolin-stimulated cAMP accumulation in rat cultured cerebellar astrocytes: L-cysteine sulfinic acid (L-CSA) = L-aspartate > L-glutamate >/= the glutamate uptake inhibitor, L-PDC. This property did not reflect activation of conventional glutamate receptors, since the selective ionotropic glutamate receptor agonists NMDA, AMPA, and kainate, as well as several mGlu receptor agonists [(1S,3R)-ACPD, (S)-DHPG, DCG-IV, L-AP4, L-quisqualate, and L-CCG-I], were without activity. In addition, the mGlu receptor antagonists, L-AP3, (S)-4CPG, Eglu, LY341495, (RS)-CPPG, and (S)-MCPG failed to reverse 30 microM glutamate-mediated inhibitory responses. L-PDC-mediated inhibition was abolished by the addition of the enzyme glutamate-pyruvate transaminase. This finding suggests that the effect of L-PDC is indirect and that it is mediated through endogenously released L-glutamate. Interestingly, L-glutamate-mediated inhibitory responses were resistant to pertussis toxin, suggesting that G(i)/G(o) type G proteins were not involved. However, inhibition of protein kinase C (PKC, either via the selective PKC inhibitor GF109203X or chronic PMA treatment) augmented glutamate-mediated inhibitory responses. Although mGlu3 receptors (which are negatively coupled to adenylyl cyclase) are expressed in astrocyte populations, in our study Western blot analysis indicated that this receptor type was not expressed in cerebellar astrocytes. We therefore suggest that cerebellar astrocytes express a novel mGlu receptor, which is negatively coupled to adenylyl cyclase, and possesses an atypical pharmacological profile.