Search PubMed⌕ Search

Biomedical subjects

L A Fitzpatrick

Publications and source records attributed to L A Fitzpatrick.

At least 109 records · Page 6Linked to original sources
PubMed1994

Direct effect of calcium channel antagonists on osteoclast function: alterations in bone resorption and intracellular calcium concentrations.

Osteoclasts attach to mineralized surfaces and resorb bone matrix, releasing calcium into the area surrounding the osteoclast. The production of high levels of extracellular calcium increases intracellular calcium concentrations ([Ca2+]i), and bone resorption is decreased. To study this mechanism, the dihydropyridine-sensitive L-type calcium channel antagonists PN 200-110, (-)202-791, and nifedipine were studied for their effects on bone resorption using the disaggregated osteoclast pit assay. Changes in [Ca2+]i after treatment with these compounds were determined with the fluoroprobe fura2. In osteoclast-enriched cultures, significant decreases in bone resorption were noted in the presence of PN 200-110 and (-)202-791. The decrease in bone resorption correlated with an increase in [Ca2+]i. To determine whether the effects of these compounds on osteoclasts were mediated via osteoblasts, proliferation and differentiation of rat osteoblast-like cells (ROS 17/2.8) were examined after the addition of these agents. There were no changes in osteoblast proliferation or differentiation, as determined by [3H]thymidine incorporation and specific activity of alkaline phosphatase, after treatment with these compounds at concentrations that inhibited bone resorption in the disaggregated pit assay. This lack of effect of calcium channel antagonists on osteoblast growth and differentiation at concentrations used to inhibit osteoclast function suggests that the effects of PN 200-110 and (-)202-791 on the osteoclast are not mediated via the osteoblast. In addition, conditioned medium recovered from ROS 17/2.8 cultures treated with PN 200-110 or (-)202-791 had no effect on pit formation compared to the conditioned medium from cell-free cultures. This lack of effect of calcium channel conditioned medium on bone resorption provides additional evidence that PN 200-110 and (-)202-791 are decreasing bone resorption directly by altering osteoclast function, not through osteoblast-osteoclast interactions. The addition of (-)202-791 or PN 200-110 to osteoclasts resulted in a dose-dependent rise in [Ca2+]i. These data suggest that calcium channel antagonists may bind to the calcium channel of the osteoclast and lock it in an open state, leading to increased [Ca2+]i and decreased bone resorption.

Animals↗
PubMed1994

Secretion of insulin-like growth factor binding protein-1 from individual hepatocytes.

The reverse hemolytic plaque assay (RHPA) uses complement-mediated red blood cell lysis to detect peptide secretion by individual cells. Initially, the RHPA was used to study the function of neurons and B lymphocytes. More recently, the RHPA has been adapted to measure hormone release from individual pituitary, parathyroid, luteal, and pancreatic islet cells. We have applied this technique to detect insulin-like growth factor binding protein-1 (IGFBP-1) secretion by a human hepatoma cell line (HepG2). We proposed that the technique of RHPA could be used to study peptide release from single hepatocytes in various defined conditions. Our goal was the study of the kinetics of IGFBP-1 secretion from hepatoma cells and rat hepatocytes and to determine the heterogeneity of the cell population regarding the secretion of IGFBP-1. To evaluate the optimal conditions of IGFBP-1 secretion by hepatoma cells and rat hepatocytes and to evaluate the influence of cell dispersion on hepatocyte's behavior, we evaluated three techniques of cell dispersion: trypsin digestion, collagenase digestion, and mechanical dispersion. We tested cell viability, determined the percentage of secreting cells versus non-secreting cells, and measured mean plaque area which is a function of the amount of IGFBP-1 secreted by an individual cell. We determined the optimal IGFBP-1 antibody dilution for the detection of secreted IGFBP-1 by hepatocytes, evaluated the initiation of IGFBP-1 secretion from cultured cells, and quantified time-dependent IGFBP-1 secretion. In addition to demonstrating the feasibility of measuring IGFBP-1 from a cultured cell line, we measured IGFBP-1 release from freshly dispersed rat hepatocytes.

Animals↗
PubMed1993

Distribution of noncollagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity.

The microanatomic distribution of several noncollagenous proteins (NCPs) in bone matrix was examined by immunohistochemical analysis of glycol-methyl methacrylate-embedded normal adult human bone biopsies. Osteopontin and bone sialoprotein stained throughout the lamellae of both trabecular and cortical bone. Cement lines (cortical and trabecular) and the mineralized matrix immediately adjacent to each Haversian canal were intensely stained. Osteocalcin was detected in cement lines; however, lamellar staining varied depending on the location within the individual unit of bone. In cortical bone, the inner concentric lamellae of osteons were often unstained but the outer lamellae were heavily stained for osteocalcin. Osteonectin was not detected in cement lines and in most specimens revealed a pattern similar to that of osteocalcin with respect to the absence of immunostaining within the inner concentric lamellae. Decorin was prominent in the perilacunar matrix, the canaliculi of osteocytes, and the matrix immediately adjacent to quiescent Haversian canals. Biglycan appeared evenly distributed throughout cortical and trabecular bone matrix. These results suggest that the incorporation of NCPs into matrix may vary depending on the stage of formation of individual bone units. The specific distribution and spatial relationship of these NCPs may be related to the function of each protein during bone resorption and formation. The distinct patterns of NCP localization in bone support the hypothesis that in addition to their structural and mineral-inducing properties, these proteins may influence the events associated with bone remodeling, such as recruitment, attachment, differentiation, and activity of bone cells.

Adult↗
PubMed1993

Noninvasive testing in the diagnosis of osteomalacia.

PURPOSE: Osteomalacia is associated with many clinical, radiographic, and biochemical abnormalities. Unfortunately, none of these are pathognomonic of the disorder, and histologic examination of a bone biopsy specimen is often necessary to confirm the diagnosis. Noninvasive methods of diagnosis would be preferable to decrease patient morbidity and increase cost-effectiveness. Previous studies addressing the adequacy of these methods were performed prior to the widespread availability of 1,25-dihydroxycalcitriol (1,25-(OH)2D3) and parathyroid hormone (PTH) levels. Therefore, we reviewed our experience with patients with biopsy-proven osteomalacia to determine if PTH or 1,25(OH)2D3 levels would serve a useful role in establishing the diagnosis of osteomalacia. METHODS: We retrospectively studied 17 patients who had biopsy-proven osteomalacia (defined as an osteoid volume greater than 10% and an osteoid width greater than 15 microns) in order to ascertain if their clinical presentation, biochemical profile, and radiographic features were sufficient to establish a diagnosis of osteomalacia. RESULTS: We found that 94% of our patients exhibited symptoms of osteomalacia, and all patients had at least one demonstrable sign of osteomalacia on examination. Biochemically, the patients presented a heterogeneous picture: 94% had an elevated alkaline phosphatase level; 47% had either a low serum calcium or phosphate (12% had both) level; urinary calcium excretion was low in 18%; and 25-(OH)D3 concentration was low in 29%. Levels of 1,25-(OH)2D3 were measured in eight patients; three had low values despite normal 25-(OH)D3 levels. PTH levels were elevated in 41% of patients in the absence of histologic evidence of hyperparathyroidism; however, these patients were noted to have multiple other abnormal clinical, biochemical, or radiograph features. Radiographically, 18% of patients had pseudofractures; the rest had nonspecific findings suggestive of osteomalacia. In summary, all patients had at least two of the following abnormalities: low calcium, low phosphate, elevated alkaline phosphatase, or a radiographic finding suggestive of osteomalacia. CONCLUSION: We conclude: (1) a careful history and physical examination remain important in the evaluation of potential osteomalacia patients; (2) PTH offered no apparent benefit as a screening test in our patients; (3) 1,25-(OH)2D3 was low in three patients with normal levels of 25-(OH)D3; (4) decreased urinary calcium excretion had low sensitivity for detecting osteomalacia; and (5) serum calcium, phosphate, alkaline phosphatase, and radiographic examination may be adequate screening tests in patients who have a clinical presentation suggestive of osteomalacia.

Alkaline Phosphatase↗
PubMed1993

Detection of subtle aluminum-related renal osteodystrophy.

We compared the sensitivity of aurin tricarboxylic acid (ATA) or acid solochrome azurine (ASA) for detecting bone aluminum histochemically in 87 biopsy specimens obtained between 1983 and 1987 from 84 patients receiving dialysis therapy. Two consecutive biopsy sections were stained, one with ATA and the other with ASA, and then interpreted independently by two experienced observers. Three groups were established: group 1 (N = 61) had positive results of both ATA and ASA staining, group 2 (N = 25) had negative ATA but positive ASA sections, and group 3 (N = 1) had negative results of both ATA and ASA. No significant differences existed between groups 1 and 2 for age of the patients or serum calcium or immunoreactive parathyroid hormone levels. Patients in group 1 had significantly higher bone aluminium content (110 versus 61 micrograms/g dry ash weight), higher serum aluminum levels (151 versus 26 ng/ml), and longer duration of dialysis (85 versus 30 months) than did patients in group 2. Bone biopsy diagnoses (group 1 versus group 2) included low-turnover bone disease, 8 versus 7; osteomalacia, 26 versus 0; mixed uremic bone disease, 10 versus 1; hyperparathyroidism, 12 versus 14; and mild uremic bone disease, 5 versus 4. On the basis of ATA staining, 7 of 15 patients with low-turnover and 1 of 11 patients with mixed uremic bone disease may have been incorrectly diagnosed as having non-aluminum-related bone disorders. The levels of bone and serum aluminum were lower in group 2 than in group 1 but still much higher than normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum↗
PubMed1993

Heterogeneous response to calcium by individual parathyroid cells.

Classical stimulus-secretion theory suggests that each individual cell responds to a given stimulus. We tested this theory by determining the response of single bovine parathyroid cells to calcium with the reverse hemolytic plaque assay (RHPA), an assay that measures hormone release from individual cells. As calcium concentrations decreased, the amount of parathyroid hormone (PTH) released per cell increased, and cells were recruited to release PTH. To confirm that adequate stores of PTH were present, immunocytochemistry and in situ hybridization were performed. To test if cells that did not release PTH were capable of secretion, we performed a sequential RHPA; 47.8% of cells did not release PTH after the first stimulus. After the second exposure to low concentrations of calcium, 26.5% of these "nonsecretory" cells were able to release PTH. We conclude that parathyroid cells are homogeneous for PTH content and synthetic capability. Parathyroid cells respond to changes in extracellular calcium heterogeneously in that more PTH per cell is released, and individual parathyroid cells are "recruited" to release PTH at low calcium concentrations. In addition, parathyroid cells can be induced to secrete suggesting that cells are viable but in a depressed secretory state. Parathyroid cells may exist in an "on" or "off" secretory state.

Adrenocorticotropic Hormone↗
PubMed1993

Regulation of parathyroid hormone release by protein kinase-C is dependent on extracellular calcium in bovine parathyroid cells.

The purpose of this study was to evaluate regulation of PTH secretion by protein kinase-C (PKC) in adult bovine parathyroid cells. Extracellular calcium (Ca2+e) is the main physiological regulator of PTH secretion. Putative second messengers include intracellular calcium (Ca2+i), cAMP, inositol trisphosphate, and diacylglycerol (DAG). Both DAG and Ca2+i activate PKC. Certain phorbol esters mimic the effect of DAG and cause prolonged stimulation of PKC. The stimulatory phorbol esters 12-O-tetradecanoylphorbol acetate (1 microM) and phorbol-12,13-dibutyrate (1 microM) did not affect PTH secretion at low Ca2+e, but increased both individual cell secretion and recruitment of cells to secrete at high Ca2+e. The PKC inhibitors H7 (1 microM), tamoxifen (10 microM), and sphinganine (5 microM) inhibited PTH release at low Ca2+e (0.1 and 0.2 mM) and decreased cell recruitment over the physiological range of Ca2+e. The nonstimulatory phorbol esters 4 alpha-phorbol-12,13-didecanoate (1 microM) and phorbol-13-monoacetate (1 microM) had no effect on PTH secretion. To assess the mechanism by which certain phorbol esters stimulated PTH secretion, in situ hybridization for PTH mRNA was performed. Phorbol-12,13-dibutyrate (1 microM) qualitatively increased steady state PTH mRNA levels compared to control values. We conclude that 1) PKC stimulation increased PTH secretion at high Ca2+e, but not at low Ca2+e; 2) PKC inhibition decreased PTH secretion at low Ca2+e; and 3) PKC stimulation increased steady state PTH mRNA levels. These data suggest that PKC plays an important regulatory role in the synthesis and secretion of PTH.

Animals↗
PubMed1993

Hormone secretion by normal human parathyroid cells in vitro: characterization by the reverse hemolytic plaque assay.

We have recently validated a reverse hemolytic plaque assay (RHPA) for the quantitative measurement of parathyroid hormone release from single isolated parathyroid cells. The information regarding secretory behavior of normal and abnormal parathyroid tissue has been based previously on fluorescent spectroscopy or quantification of immunoreactive parathyroid hormone by radioimmunoassay. Thus, individual differences in secretion and assessment of viability are not clearly measured in these types of experiments. The RHPA has the advantage of allowing measurements of secretory activity in individual viable parathyroid cells. In this report, we have modified the RHPA to measure the release of parathyroid hormone from human cells. This is the first instance of measurement of hormone secretion from human cells, and we present data indicating the specificity of the assay for human parathyroid hormone. The response of normal human parathyroid cells to alterations in extracellular calcium was assessed by two parameters: the ability of an individual cell to secrete parathyroid hormone in response to a stimulus and the recruitment of normal human cells to release parathyroid hormone. This assay will allow elucidation of secretory dynamics in normal and abnormal glands, which may help us to understand the mechanisms involved in parathyroid dysfunction.

Animals↗
PubMed1992

Parathyroid autotransplantation.

Although rare following initial cervical exploration, reoperative parathyroid surgery may cause permanent hypoparathyroidism in 15% to 30% of patients. Immediate fresh or delayed cryopreserved parathyroid autotransplantation is the principal surgical option to resolve this complication. Between 1980 and 1990, 18 and 12 patients underwent immediate and cryopreserved autotransplantation, respectively. With a mean follow-up of nearly 5 years, 61% of the immediate and 42% of the cryopreserved tissue showed evidence of function. However, only 10 (55%) and two (17%) of the respective patients had completely discontinued treatment with calcium supplementation. Graft-dependent hypercalcemia can occur with either technique. We conclude that until cryopreserved tissue can be transplanted with more reliable success and if the excised abnormal parathyroid likely represents the only remaining gland, we would advise immediate autotransplantation. In the reoperative setting, unless a residual normal parathyroid gland is confirmed, a portion of the excised parathyroid tissue should be cryopreserved for possible autotransplantation in case hypoparathyroidism develops subsequently.

Cryopreservation↗
PubMed1992

Chromogranin-A secretion from individual parathyroid cells: effects of 1,25-(OH)2 vitamin D3 and calcium.

Chromogranin-A (CgA) is a 50-kDa protein located in and secreted by most endocrine and neuroendocrine cells along with native hormone. In the parathyroid gland, CgA is cosecreted with parathyroid hormone (PTH). Although these peptides are secreted together, recent evidence has suggested that they are processed differently in response to stimuli, such as 1,25-(OH)2 vitamin D3 and calcium. We have validated a reverse hemolytic plaque assay for studying CgA release from parathyroid cells. This assay allows for the detection of quantitative changes in hormone secretion from individual parathyroid cells. Bovine parathyroid cells were mixed with protein-A-linked ovine erythrocytes (oRBC) and plated in a monolayer in the presence of CgA antiserum. After incubation, complement was added to the cells to induce cell lysis. Lysis of oRBC around a parathyroid cell indicated the release of CgA from a cell. Results showed that plaque formation was dependent on assay reagents and that serial dilution of the antibody reduced plaque formation. CgA secretion was inhibited by increasing concentrations of calcium and stimulated by increasing concentrations of 1,25-(OH)2vitamin D3.

Animals↗
PubMed1992

Osteoporosis and calcification of the aorta.

In an age-stratified random sample of 200 Rochester, Minnesota women, the prevalence of aortic calcification rose with aging, as did the prevalence of vertebral fractures, while bone mass fell. The statistically significant positive association of aortic calcification with vertebral fractures and the negative associations with bone mass at six skeletal sites were mainly accounted for by age. After age-adjustment, the only association remaining was a negative one between calcified aortic plaques and bone mineral density (BMD) of the lumbar spine (P < 0.05). Aortic calcification was not associated with any measures of calcium metabolism, after adjusting for age, except for a slight negative association between linear aortic calcifications and 25(OH) vitamin D levels (P < 0.05). BMD values of the lumbar spine were somewhat greater than predicted for age in women with severe aortic calcification, but similar findings were seen at other skeletal sites and none of the differences was statistically significant. While overestimation of bone mass was generally minimal, severe aortic calcification may distort lumbar spine assessments in a minority of postmenopausal women.

Adult↗
PubMed1992

Individual parathyroid cells exhibit cyclic secretion of parathyroid hormone and chromogranin-A (as measured by a novel sequential hemolytic plaque assay).

PTH and chromogranin-A (CgA) are the two major proteins secreted from the parathyroid gland. We investigated the secretory patterns of CgA and PTH using a sequential reverse hemolytic plaque assay (RHPA). The RHPA allows detection of hormone secretion from individual cells after a secretory stimulus. For the sequential RHPA, bovine parathyroid cells were mixed with protein-A-conjugated ovine erythrocytes (oRBC). Antiserum, either anti-PTH or anti-CgA, was added under optimal secretory conditions. The addition of complement caused lysis of oRBC surrounding hormone-secreting cells. In this stage (stage 1), individual cells were identified and indexed as secreting or nonsecreting cells. For stage 2, a new lawn of oRBC was established, and a second RHPA was performed on the same population of cells, allowing for the detection of secretory patterns. In the single stage RHPA, about three fourths of the cells formed CgA plaques compared to only about half that formed PTH plaques, suggesting that CgA and PTH are not always cosecreted. In the sequential RHPA, of the cells that did not secrete CgA or PTH in stage 1, up to half secreted in stage 2. Of those cells that secreted in stage 1, up to one fourth did not secrete in stage 2. These results indicate that the parathyroid cells "cycled" between secretory and nonsecretory phases. Our experimental design precluded our obtaining unequivocal data on whether CgA is an autocrine/paracrine regulator of PTH secretion.

Animals↗
PubMed1992

Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific.

Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.

Animals↗
PubMed1992

The effect of cryopreservation on cell viability and hormone secretion in human parathyroid tissue.

BACKGROUND: To explain the decreased success of cryopreserved versus fresh parathyroid autotransplants, we investigated the effect of cryopreservation on parathyroid function. METHODS: Parathyroid cryopreserved tissue from surgical specimens of 18 patients was analyzed in a comparative fashion with 10 fresh abnormal glands. Cell viability was evaluated by a double-fluorescence technique, and parathyroid hormone secretion was detected from individually dispersed parathyroid cells with the reverse hemolytic plaque assay. The plaque area reflected the amount of parathyroid hormone secreted by each cell, and the percentage of plaque-forming cells represented the number of secreting cells. Feedback regulatory mechanisms remained intact for both fresh and cryopreserved tissue as shown over a wide range of calcium concentrations. RESULTS: The percentage of viable cells from fresh and cryopreserved tissue was always in excess of 88%. No significant difference was noted in the percentage of plaque-forming cells nor the amount of hormone released per individual cell comparing fresh and cryopreserved glands (6 months to 2 years). Specifically, at physiologic calcium concentrations no significant differences existed in the secretory behavior between fresh and cryopreserved tissue. CONCLUSIONS: Without showing decreased viability or parathyroid hormone secretion after cryopreservation, the cause of graft failure in cryopreserved tissue still remains unknown.

Calcium↗
PubMed1992

Parathyroid carcinoma: clinical and pathologic features in 43 patients.

Parathyroid carcinoma accounts for 0.5 to 5% of all cases of hyperparathyroidism. We reviewed the clinical, surgical, and pathologic features observed in all patients with parathyroid carcinoma evaluated at the Mayo Clinic from 1920 through 1991. Forty-three patients (22 women, 21 men; mean age, 54 yrs, range 29-72) were identified, including 2 with familial hyperparathyroidism. Information on initial presentation was available in 40 patients: 15 (38%) presented with polydipsia or polyuria, 11 (27%) with myalgias or arthralgias, 7 (17%) with weight loss, and 4 (10%) with nephrolithiasis; 3 patients (7%) were asymptomatic at presentation. Of 31 patients in whom the initial neck examination was recorded, 14 (45%) had a palpable neck mass. The mean serum calcium and serum phosphorus levels were 14.6 mg/dl and 2.3 mg/dl, respectively. Parathyroid hormone levels were elevated in 21 of 21 patients (mean elevation, 10.2 times upper limit of normal). Complications included nephrolithiasis in 14 of 25 patients (56%), bone disease in 20 of 22 patients (91%) and both in 8 of 15 patients (53%). All patients underwent primary surgical resection of parathyroid carcinoma. Twenty-six of 43 patients (60%) required a second operation with 18 patients requiring multiple re-explorations. At the second operation, residual tumor was found in the neck (68%), mediastinum (16%), or both (12%). Six patients received radiation therapy to the neck (5 patients) or bones (1 patient) for recurrent or metastatic disease. Of these, 1 patient appeared cured of parathyroid carcinoma by radiation therapy 11 years after documented tumor invasion of his trachea. Repeated excision of tumor recurrences was an effective means of controlling hypercalcemia in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Academic Medical Centers↗