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R T Jensen

Publications and source records attributed to R T Jensen.

At least 91 records · Page 5Linked to original sources
PubMed1998

Preoperative diagnosis and surgical management of neuroendocrine gastroenteropancreatic tumors: general recommendations by a consensus workshop.

In 1996 a consensus workshop on the preoperative diagnosis and surgical treatment of neuroendocrine tumor disease took place in Berlin. Although there was a consensus on the diagnosis and surgical treatment by world experts in neuroendocrine surgery in some areas, it became clear that certain management policies vary among centers. In large part, diverging policies reflect a lack of controlled studies. This paper summarizes the various opinions brought forward during this conference and emphasizes consensus approaches for the diagnosis and therapy of neuroendocrine neoplasms.

Gastrointestinal Neoplasms↗
PubMed1998

Effect of long-term gastric acid suppressive therapy on serum vitamin B12 levels in patients with Zollinger-Ellison syndrome.

BACKGROUND AND AIMS: Long-term treatment with H(+)-K(+)-adenotriphosphatase (ATPase) inhibitors, such as omeprazole or lansoprazole, for severe gastroesophageal reflux disease is now widely used. Whether such treatment will result in vitamin B12 deficiency is controversial. We studied whether long-term treatment with omeprazole alters serum vitamin B12 levels in patients with Zollinger-Ellison syndrome. METHODS: In 131 consecutive patients treated with either omeprazole (n = 111) or histamine H2-receptor antagonists (n = 20), serum vitamin B12 and folate levels and complete blood counts were determined after acid secretion had been controlled for at least 6 months. These studies were repeated yearly. Serum vitamin B12 and folate levels were correlated with the type of antisecretory drug and the extent of inhibition of acid secretion. RESULTS: The mean duration of omeprazole treatment was 4.5 years, and for H2-receptor antagonists 10 years. Vitamin B12 levels, but not serum folate levels or any hematological parameter, were significantly (P = 0.03) lower in patients treated with omeprazole, especially those with omeprazole-induced sustained hyposecretion (P = 0.0014) or complete achlorhydria (P < 0.0001). In 68 patients with two determinations at least 5 years apart, vitamin B12 levels decreased significantly (30%; P = 0.001) only in patients rendered achlorhydric. The duration of omeprazole treatment was inversely correlated with vitamin B12 levels (P = 0.013), but not folate levels. Eight patients (6%) developed subnormal B12 levels during follow-up. CONCLUSIONS: Long-term omeprazole treatment leads to significant decreases in serum vitamin B12 but not folate levels. These results suggest patients with Zollinger-Ellison syndrome treated with H(+)-K(+)-ATPase inhibitors should have serum vitamin B12 levels monitored. Furthermore, these results raise the possibility that other patients treated chronically with H(+)-K(+)-ATPase inhibitors may develop B12 deficiency.

Achlorhydria↗
PubMed1998

A human gene encodes a putative G protein-coupled receptor highly expressed in the central nervous system.

The mammalian bombesin (Bn)-like neuropeptide receptors gastrin-releasing peptide receptor (GRP-R) and neuromedin B receptor (NMB-R) transduce a variety of physiological signals that regulate secretion, growth, muscle contraction, chemotaxis and neuromodulation. We have used reverse transcription-polymerase chain reaction (PCR) to isolate a cDNA from human brain mRNA, GPCR/CNS, that encodes a putative G protein-coupled receptor (GPCR) based upon the presence of the paradigmatic seven heptahelical transmembrane domains in its predicted amino acid sequence. Analysis of the deduced protein sequence of GPCR/CNS reveals this putative receptor to be 98% identical to the deduced amino acid sequence of a recently reported gene product and minimally identical (approximately 23%) to both murine GRP-R and human endothelin-B (ET-B) receptor. Our deduced protein sequence differs at 12 positions, scattered throughout the open reading frame, relative to the original sequence. A 3.7 kb GPCR/CNS mRNA species is expressed in vivo in a tissue-specific manner, with highest levels detected in brain and spinal cord, lower levels found in testis, placenta and liver, but no detectable expression observed in any other tissue. Analysis of GPCR/CNS genomic clones reveals that the human gene contains one intron that is about 21 kb in length that divides the coding region into two exons and maps to human chromosome 7q31. No specific binding is observed with either a newly identified ligand (DTyr6, beta Ala11, Phe13, Nle14]Bn-(6-14)) having high affinity for all Bn receptor subtypes or Bn after GPCR/CNS is stably expressed in fibroblasts. No elevation in inositol trisphosphate is observed after the application of micromolar levels of either DPhe6, beta Ala11, Phe13, Nle14]Bn-(6-14) or Bn, a concentration of agonist known to activate all four known Bn receptor subtypes. When GPCR/CNS is expressed in Xenopus oocytes, no activation of the calcium-dependent chloride channel is detected despite the addition of micromolar levels of Bn peptide agonists. We conclude that the natural ligand for this receptor is none of the known naturally occurring Bn-like peptides and the true agonist for GPCR/CNS remains to be elucidated.

3T3 Cells↗
PubMed1998

Iron absorption in patients with Zollinger-Ellison syndrome treated with long-term gastric acid antisecretory therapy.

BACKGROUND: Gastric acid secretion is important for absorption of dietary non-haem iron, and iron deficiency is common in gastric hyposecretory states such as after gastric resection. It is not known if prolonged, continuous treatment with potent acid suppressants such as omeprazole will lead to iron deficiency or lower body iron stores. AIM: To assess iron stores and the occurrence of iron deficiency anaemia in patients with Zollinger-Ellison syndrome (ZES) treated long-term with gastric antisecretory drugs. METHODS: One hundred and nine patients with ZES but without previous gastric resections were studied. All patients underwent assessment of acid control on antisecretory agents, determination of tumour extent, evaluation of haematological parameters (Hct, haemoglobin, WBC, MCV, MCHC), and determination of serum iron parameters (iron, ferritin, transferrin, iron/ transferrin ratio). Acid control values for the last 4 years were reviewed, the presence or absence of acid hyposecretion determined using three different criteria and this result correlated with haematological and iron parameters. RESULTS: Eighty-nine patients were taking omeprazole, nine patients were taking histamine H2-antagonists and 11 patients were taking no drugs following curative resection. The mean duration of omeprazole treatment was 5.7 years (range 0.7-12.5 years) and total duration of any treatment was 10.1 years (range 0.7-21 years). Acid hyposecretion was present by at least one criterion in 45% of patients. There were no significant differences between patients with or without acid hyposecretion, taking or not taking omeprazole, having different durations of omeprazole treatment or different durations of total time receiving any antisecretory treatment, for any serum iron parameter, haematological parameter, or for the frequency of iron deficiency. Males and females did not differ in percentage with low ferritin levels or percentage with iron deficiency. CONCLUSIONS: Continuous treatment with omeprazole for 6 years or continuous treatment with any gastric antisecretory drug for 10 years does not cause decreased body iron stores or iron deficiency. These results suggest that, in contrast to recent results which show yearly monitoring of vitamin B12 in such patients is needed, such monitoring for iron parameters is not necessary.

Adolescent↗
PubMed1998

Management of the Zollinger-Ellison syndrome in patients with multiple endocrine neoplasia type 1.

Zollinger-Ellison syndrome (ZES) is the most common symptomatic pancreatic endocrine tumour in patients with MEN-1. Besides the treatment of the usual endocrinopathies seen in patients with MEN-1, the treatment of the ZES requires attention be paid to controlling the gastric acid hypersecretion, to dealing with the gastrinomas per se which are malignant in 18-60% of cases, and to the diagnosis and treatment of gastric carcinoid tumours, that are increasingly seen in these patients. In this article the current management of each of the areas is reviewed and what is known or uncertain discussed, based on our studies at the NIH and data from others. Data from 231 patients including 45 with MEN-1 and 186 without MEN-1 is contrasted in this report. Gastric acid hypersecretion has been controlled in all patients medically with MEN-1 and ZES at the NIH for up to 22 years. The current drugs of choice are H+-K+ ATPase inhibitors and twice a day dosing is recommended. Periods of parenteral drug therapy (surgery, etc.) and pregnancy require important modifications. The appropriate surgical therapy of the gastrinoma is controversial. Eighty per cent of patients have a duodenal gastrinoma and 20-30% have a pancreatic tumour. Recent studies suggest gastrinoma enucleation combined with duodenotomy rarely results in cure. Aggressive surgery (Whipple resection) can result in cure of gastrinoma but effect on survival is unclear. There are important differences in gastrinoma location, extent, and percentage with aggressive disease in patients with or without MEN-1, which are discussed. Confusion has occurred because of lack of information on the natural history of the gastrinoma compared to the other pancreatic endocrine tumours that occur in MEN-1 and survival data from patients with and without MEN-1 is contrasted. The occurrence of gastric carcinoids in patients with and without MEN-1 with ZES is contrasted and the areas of certainty and disagreement reviewed.

Humans↗
PubMed1998

Analysis of factors associated with long-term (five or more years) cure in patients undergoing operation for Zollinger-Ellison syndrome.

BACKGROUND: Only 30% to 40% of patients with Zollinger-Ellison syndrome (ZES) undergoing operation with curative intent have biochemical cures. The purpose of this analysis was to identify perioperative factors associated with long-term (> or = 5 years) biochemical cures. METHODS: From December 1981 to September 1997, 128 patients with potentially curable ZES underwent 152 abdominal explorations with resection of all identifiable disease. Of these, 31 outcomes were identified with documented cures at > or = 5 years from operation and were compared with outcomes of 110 patients who were not cured. Univariate and multivariate statistical analyses were performed. RESULTS: The results of a normal immediate postoperative fasting serum gastrin and secretin stimulation test were significantly and independently correlated with 5-year cure (P2 = .005 and .0099, respectively). A diagnosis of multiple endocrine neoplasia type 1 was significantly inversely correlated with cure on univariate analysis (P2 = .027). Gender, age, duration of symptoms, results of diagnostic tests, or results of imaging studies did not correlate with outcome. CONCLUSIONS: All patients with sporadic potentially curable ZES should undergo exploration because outcome is not associated with preoperative tests. Only 5% of patients with ZES and multiple endocrine neoplasia type 1 were cured at 5 years. A normal fasting serum gastrin or secretin stimulation value value immediately postoperatively provides important prognostic information.

Female↗
PubMed1998

Operative management of islet-cell tumors arising in the head of the pancreas.

BACKGROUND: Pancreatic islet cell tumors (ICTs) can be treated by enucleation or pancreatic resection. We reviewed our experience with ICTs in the head of the pancreas to define indications for enucleation versus pancreaticoduodenectomy. METHODS: Between January 1982 and December 1997, 48 patients underwent surgical resection for presumed ICTs of the pancreatic head. Of these, 18 were found on pathologic examination to be disease in a lymph node. Thirty patients had 32 true pancreatic head ICTs. We reviewed the operative results and postoperative courses in these patients. RESULTS: Mean diameter of the ICTs was 15 mm. Twenty-seven patients (90%) underwent successful enucleations. Three patients failed enucleation and underwent pancreaticoduodenectomy. There was no operative mortality. The median time to regular diet was 7 days. The median time to removal of all drains was 22 days. The most common complication was pancreatic fistula (15%). No patient required reoperation for treatment of a fistula. CONCLUSION: Most ICTs of the pancreatic head can be removed with enucleation, preserving pancreatic tissue and avoiding the morbidity of pancreaticoduodenectomy. The primary indication for pancreaticoduodenectomy is not the size of the lesion but its proximity to the pancreatic duct.

Adolescent↗
PubMed1998

Surgical treatment of localized gastrinoma within the liver: a prospective study.

BACKGROUND: Studies demonstrate that liver metastases of gastrinoma significantly reduce survival. METHODS: Since 1982 we have prospectively studied 213 patients with Zollinger-Ellison syndrome. For this report the results of surgery for localized liver gastrinoma were analyzed. RESULTS: Zollinger-Ellison syndrome was diagnosed biochemically in all patients and acid output was controlled with medications. Imaging studies demonstrated liver gastrinoma in 69 patients (32%). Fifty-two had diffuse unresectable disease, whereas 17 (10%) had localized disease. All patients with localized liver gastrinoma and 2 patients with diffuse disease who needed surgery are the subject of this report. Major hepatic lobectomy was performed in 10 patients and wedge resections in 9. Three patients had apparent liver primary gastrinomas and 16 had metastatic disease. Seventeen of 19 patients were able to have all identifiable gastrinoma resected. Extrahepatic tumor was also removed at the same procedure. Extirpation of liver gastrinoma required hepatic lobectomy in 10 patients and wedge resections in the others. Five-year survival was 85%. Five of 17 completely resected patients (29%) remained disease free. CONCLUSIONS: Resectable localized liver gastrinoma is rare. Primary liver gastrinomas can occur. Surgical resection of localized liver gastrinoma provides a cure rate similar to that of extrahepatic gastrinoma and an excellent long-term survival.

Female↗
PubMed1998

Prospective study of somatostatin receptor scintigraphy and its effect on operative outcome in patients with Zollinger-Ellison syndrome.

OBJECTIVE: To determine the relative abilities of somatostatin receptor scintigraphy (SRS) and conventional imaging studies (computed tomography, magnetic resonance imaging, ultrasound, angiography) to localize gastrinomas before surgery in patients with Zollinger-Ellison syndrome (ZES) subsequently found at surgery, and to determine the effect of SRS on the disease-free rate. SUMMARY BACKGROUND DATA: Recent studies demonstrate that SRS is the most sensitive imaging modality for localizing neuroendocrine tumors such as gastrinomas. Because of conflicting results in small series, it is unclear in ZES whether SRS will alter the disease-free rate, which gastrinomas are not detected, what factors contribute to failure to detect a gastrinoma, or whether the SRS result should be used to determine operability in patients without hepatic metastases, as recently recommended by some investigators. METHODS: Thirty-five consecutive patients with ZES undergoing 37 exploratory laparotomies for possible cure were prospectively studied. All had SRS and conventional imaging studies before surgery. Imaging results were determined by an independent investigator depending on surgical findings. All patients underwent an identical surgical protocol (palpation after an extensive Kocher maneuver, ultrasound during surgery, duodenal transillumination, and 3 cm duodenotomy) and postoperative assessment of disease status (fasting gastrin, secretin test imaging within 2 weeks, at 3 to 6 months, and yearly), as used in pre-SRS studies previously. RESULTS: Gastrinomas were detected in all patients at each surgery. Seventy-four gastrinomas were found: 22 duodenal, 8 pancreatic, 3 primaries in other sites, and 41 lymph node metastases. The relative detection order on a per-patient or per-lesion basis was SRS > angiography, magnetic resonance imaging, computed tomography > ultrasound. On a per-lesion basis, SRS had greater sensitivity than all conventional studies combined. SRS missed one third of all lesions found at surgery. SRS detected 30% of gastrinomas < or =1.1 cm, 64% of those 1.1 to 2 cm, and 96% of those >2 cm and missed primarily small duodenal tumors. Tumor size correlated closely with SRS rate of detection. SRS did not increase the disease-free rate immediately after surgery or at 2 years mean follow-up. CONCLUSIONS: SRS is the most sensitive preoperative imaging study for extrahepatic gastrinomas in patients with ZES and should replace conventional imaging studies as the preoperative study of choice. Negative results of SRS for localizing extrahepatic gastrinomas should not be used to decide operability, because a surgical procedure will detect 33% more gastrinomas than SRS. SRS does not increase the disease-free rate. In the future, more sensitive methods to detect small gastrinomas, especially in the duodenum and in periduodenal lymph nodes, or more extensive surgery will be needed to improve the postoperative disease-free rate in ZES.

Adult↗
PubMed1998

Bone metastases in patients with gastrinomas: a prospective study of bone scanning, somatostatin receptor scanning, and magnetic resonance image in their detection, frequency, location, and effect of their detection on management.

PURPOSE: To determine whether bone scan, magnetic resonance imaging (MRI), or somatostatin receptor scintigraphy (SRS) is best for identifying bone metastases in patients with gastrinomas, as well as their frequency and location, whether their detection affects management, and what patient subgroups should be examined. MATERIALS AND METHODS: One hundred fifteen patients with gastrinoma were prospectively studied. Patients were examined yearly and those with liver metastases were reexamined every 3 months. Based on clinical history, histology, growth pattern, and development of new bone lesions, possible bone metastases were classified as to whether they were or were not bone metastases. Imaging results were correlated at different times in the disease course and with disease extent. RESULTS: Bone scan was positive in 52 patients, MRI in seven, and SRS in six. Eight patients (7%) were determined to have bone metastases and MRI was correctly positive in seven, SRS in six, and bone scan in five. SRS or MRI was positive in all patients with bone metastases. Bone scan had significantly lower specificity and sensitivity, and a higher rate (P < .02) of false-negative results than MRI or SRS. Bone metastases occurred in 31% of patients with liver metastases and 0% with only lymph node metastases. The initial bone metastases were in the spine or sacrum (75%) followed in descending order by the pelvis or sacroiliac joints (38%), scapula or shoulder, and ribs. In all cases, detection of bone metastases changed the management. CONCLUSION: SRS and MRI, because of high sensitivity and specificity, are recommended over bone scanning to screen for bone metastases in patients with gastrinomas. However, because bone metastases can occur initially outside the axial skeleton, SRS is the recommended initial localization method of choice. Bone metastases occur in 7% of all patients and 31% of patients with liver metastases, only occur in patients with liver metastases, are usually in the axial skeleton initially, and their detection changes management in all cases. Patients with pancreatic endocrine tumors with liver metastases should undergo SRS every 6 months to 1 year to detect bone metastases.

Adolescent↗
PubMed1998

Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells.

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.

3T3 Cells↗
PubMed1998

Human circular colonic smooth muscle cells possess active somatostatin receptors.

BACKGROUND & AIMS: Somatostatin alters in vivo colonic motility in different species including humans. Few data are available on a cellular basis that could explain the effects of somatostatin on human colon motility. To address these issues we studied the effects of somatostatin on isolated human circular colonic smooth muscle cells to establish whether its actions are directly or neurally mediated. METHODS: Circular smooth muscle cells were prepared by enzymatic digestion from surgical specimens of human colon (Sigma) and resuspended in HEPES buffer containing protease inhibitors. RESULTS: Cholecystokinin (1 nM), carbachol (30 nM) and KCl (20 mM) each caused a contraction of 17%, 16.5% and 15%, respectively. 1 microM of either somatostatin-14, somatostatin-28 or SMS 201-995 alone were able to produce a contraction of 5.1%, 5.7%, and 6.8%, respectively. When smooth muscle cells were preincubated with each of the above-mentioned somatostatin analogs, cholecystokinin-mediated contraction was dose-dependently inhibited only in the presence of antiproteases. The half-maximal effective concentration (EC50) for somatostatin-14, somatostatin-28 and SMS 201-995 were similar (3.5, 5.6, 3.2 nM, respectively). CONCLUSIONS: Somatostatin acts directly on human circular colonic smooth muscle cells through specific somatostatin receptors. SMS 201-995, a somatostatin receptor subtype-2 preferring analogue, shows a high affinity in inhibiting cholecystokinin-mediated contraction, suggesting the presence of somatostatin receptor subtype-2 on human circular colonic smooth muscle cells.

Aged↗
PubMed1997

Neuromedin B receptor activation causes tyrosine phosphorylation of p125FAK by a phospholipase C independent mechanism which requires p21rho and integrity of the actin cytoskeleton.

Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth. Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action. In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells. NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP). The stoichiometric relationships between increased p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in [Ca2+]i, but had no effect on NMB-stimulation of p125(FAK) phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [3H]inositol phosphates. Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125(FAK) completely. In contrast, colchicine, an agent which disrupts microtubules, had no effect. Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125(FAK) by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). NMB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB-induced changes in [Ca2+]i or PKC. The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK). These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth.

ADP Ribose Transferases↗
PubMed1997

Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho.

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125(FAK)) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21(rho) activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125(FAK) and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125(FAK) and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125(FAK) and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21(rho), caused significant inhibition of CCK-8-stimulated p125(FAK) and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125(FAK) and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21(rho).

Actins↗
PubMed1997

Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors.

An orphan receptor discovered in 1993 was called bombesin receptor subtype 3 (BRS-3) because of 47-51% amino acid identity with bombesin (Bn) receptors. Its pharmacology is unknown, because no naturally occurring tissues have sufficient receptors to allow studies. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the human non-small cell lung cancer cells, NCI-H1299, and the other was made in Balb 3T3 cells, which lack endogenous BRS-3. [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14) (where Nle represents norleucine) was discovered to have high potency for stimulating inositol phosphate formation in both cell lines. [125I-D-Tyr6,beta-Ala11,Phe13, Nle14]Bn-(6-14) bound to both cell lines with high affinity. Neither Bn nor 14 other naturally occurring Bn peptides bound to hBRS-3 with a Kd <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5'-(beta,gamma-imido)triphosphate inhibited binding to both cells due to a change in receptor affinity. These results demonstrate hBRS-3 has a unique pharmacology. It does not interact with high affinity with any known natural agonist or high affinity antagonist of the Bn receptor family, suggesting the natural ligand is either an undiscovered member of the Bn peptide family or an unrelated peptide. The availability of these cell lines and the hBRS-3 ligand should facilitate identification of the natural ligand for BRS-3, its pharmacology, and cell biology.

3T3 Cells↗
PubMed1997

CCK causes rapid tyrosine phosphorylation of p125FAK focal adhesion kinase and paxillin in rat pancreatic acini.

Recent studies show CCK stimulates tyrosine phosphorylation (TYR PHOSP) of a number of proteins and evidence from the pancreas and other cellular systems suggest this could be important in mediating some of CCK's growth and secretory effects. In other tissues various neuropeptides such as bombesin can cause tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) and paxillin which are important in mediating their growth effects. The purpose of the present study was to determine the effects of CCK in rat pancreatic acini on the TYR PHOSP of these latter proteins. In dispersed rat pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb (PY20) which was immunoprecipitated and then analyzed by Western blotting with anti-phosphotyrosine mAb (4G10), anti-p125FAK mAb or anti-paxillin mAb. CCK-8 at 5 min increased TYR PHOSP of five proteins of molecular weight > 60,000 including a broad M(r) 110-130,000 and M(r) 70-80,000. An increase in TYR PHOSP of both p125FAK and paxillin was detected within 1 min of adding CCK and reached a maximum at 2.5 min with a 9.1 +/- 1.9-fold increase for p125FAK and 3.6 +/- 0.6-fold for paxillin. CCK-8 caused a half-maximal increase in TYR PHOSP of p125FAK at 0.1 nM and paxillin at 0.03 nM. CCK-JMV also stimulated an increase in TYR PHOSP of both proteins, but was only 50% as efficacious as CCK-8. CCK-JMV caused a half-maximal increase at 10 nM and maximal at 1 microM for both proteins. To investigate whether the low affinity CCK receptor state also caused TYR PHOSP of both proteins, increasing concentrations of CCK-JMV were added to a maximally effective CCK-8 concentration (1 nM). Detectable inhibition of CCK-8-stimulated TYR PHOSP occurred with 1 microM CCK-JMV and with 3 microM CCK-JMV the CCK-8-stimulated response was inhibited 50% and was the same as that seen with CCK-JMV alone. These studies demonstrate that in rat pancreatic acini, CCK causes rapid TYR PHOSP of both p125FAK and paxillin. This stimulation is mediated by both the high affinity and low affinity CCK receptor states. This phosphorylation of these proteins could be important in mediating CCK's effect on the cytoskeleton or growth effects as shown for a number of other agents (oncogenes, neuropeptides, integrins).

Animals↗
PubMed1997

Identification of four amino acids in the gastrin-releasing peptide receptor that are required for high affinity agonist binding.

The bombesin family of G-protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), bombesin receptor subtype 3 (BRS-3), and bombesin receptor subtype 4 (bb4). All species homologues of GRP-R, NMB-R, and bb4 bind bombesin with dissociation constants in the nanomolar range; by comparison, human BRS-3 binds bombesin at much lower affinity (Kd >> 1 microM). We used this difference to help identify candidate residues that were potentially critical for forming the bombesin binding pocket. We reasoned that amino acids essential for bombesin binding would be conserved among all homologues of bb4, NMB-R, and GRP-R; conversely, at least one of these amino acids would not be conserved among homologues of BRS-3. Amino acid sequence alignment revealed nine residues that fit this model. We replaced each of these amino acids in mouse GRP-R with the homologous amino acid in human BRS-3. Four substitutions resulted in a significant decrease in bombesin affinity (R288H, Q121R, P199S, and A308S). The analogous mutations in BRS-3 (R127Q, H294R, S205P, and S315A) together resulted in a receptor with a 100-fold increase in bombesin and GRP affinities relative to wild-type BRS-3. From this, we propose a preliminary map of some of the amino acids comprising the agonist binding pocket.

3T3 Cells↗