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Biomedical subjects

L A Fitzpatrick

Publications and source records attributed to L A Fitzpatrick.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1991

Regulation of parathyroid hormone secretion.

Calcium is the most important physiological regulator of PTH secretion. Peak PTH secretion occurs at an intracellular calcium concentration of about 200 nM, regardless of the extracellular calcium concentration. We suggest, therefore, that intracellular calcium concentration is a regulator of PTH secretion that maintains calcium homeostasis. Other factors may be responsible for modulation of the intracellular calcium concentration, ultimately modulating PTH secretion. The "paradoxical" nature of the dependence of PTH secretion on the calcium concentration may be explained by considering PTH secretion to be unusual in a quantitative, rather than a qualitative, fashion. A possible mechanism for the control of PTH secretion by intracellular calcium, which involves calcium-activated potassium channels, is proposed. The parathyroid cell plasma membrane contains several sensors or channels by means of which the cell senses extracellular calcium. It is not clear whether these entities are coupled to each other or whether they function independently. Guanine nucleotide regulatory proteins are transducers of extracellular signals, including calcium. Several other second messengers that influence PTH secretion have also been described, but possible interactions between these messengers have not yet been determined.

Animals↗
PubMed1990

Individual parathyroid cells are more sensitive to calcium than a parathyroid cell population.

Information on the secretory behavior of individual parathyroid cells within a cell population has not previously been available. We now report a technique for examining quantitative changes in hormone secretion in individual parathyroid cells. We have used a reverse hemolytic plaque assay to measure cumulative PTH release in single isolated cells. Bovine parathyroid cells were dispersed with trypsin and mixed with staphylococcal protein-A-linked ovine erythrocytes. Cells were plated in a monolayer in the presence of PTH antiserum. After stimulation by an agonist, complement was added to the cells. Lysis of ovine erythrocytes formed a plaque around each individual cell that releases PTH. Results indicate that inhibition of PTH release by calcium was not affected by trypsinization. Plaque formation was dependent on all reagents; serial dilution of antiserum reduces plaque formation. Cells had a markedly uniform secretory response to calcium. We compared PTH release in individual cells measured by the reverse hemolytic plaque assay with hormone release in a parathyroid cell population measured by RIA. There was an inverse relationship between extracellular calcium concentrations and plaque area. Individual cells were more sensitive to calcium (ED50 = 0.4 mM Ca2+) than cell populations (ED50 = 0.8 mM Ca2+). We demonstrate that PTH release can be quantitated in single viable parathyroid cells.

Animals↗
PubMed1990

Binding of [125I]iodipine to parathyroid cell membranes: evidence of a dihydropyridine-sensitive calcium channel.

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes.

Animals↗
PubMed1990

Differences in the actions of calcium versus lanthanum to influence parathyroid hormone release.

PTH release from bovine parathyroid cells is inhibited by increasing concentrations of extracellular calcium (Ca2+). We have proposed that this inhibition is mediated by Ca2+ channels via a G-protein. To further test this hypothesis, we evaluated the effect of lanthanum (La3+), a potent Ca2+ channel antagonist that does not cross the cell membrane. PTH release was determined in dispersed bovine parathyroid cells by radioimmunoassay: extracellular Ca2+ concentration was 0.2 mM. PTH release was inhibited by maximal concentrations of La3+ to a greater extent than by Ca2+: 93% inhibition by La3+ vs. 40% by Ca2+. La3+ was more potent (set-point = 0.12 mM) than Ca2+ (set-point = 1.2 mM). Incubation of parathyroid cells with pertussis toxin, which inactivates a G-protein(s) and blocks inhibition by Ca2+, did not block the inhibition of PTH release by La3+ at the concentrations tested. The Ca2+ ionophore A23187, which potentiates the effect of Ca2+, did not enhance the inhibition of PTH release by La3+. Increasing concentrations of calcium enhanced the inhibition of PTH release by the Ca2+ channel agonist, (+)202-791. The Ca2+ channel antagonist, (-)202-791, shifted the Ca2+ inhibition curve to the right. La3+ did not alter the inhibition of PTH release by the Ca2+ channel agonist but blocked the stimulatory effect of the Ca2+ channel antagonist, (-)202-791. In summary: 1) La3+, which blocks Ca2+ channels and does not cross cell membranes, effects a greater inhibition of PTH release than Ca2+; 2) La3+, like Ca2+, overrides the effect of Ca2+ channel antagonist (-)202-791; and 3) La3+, unlike Ca2+, inhibits PTH release by a mechanism that is independent of a pertussis toxin-sensitive G-protein. There may be two cell surface sites that recognize La3+ and Ca2+ independently.

Animals↗
PubMed1989

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator.

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.

Animals↗
PubMed1989

Hypercalcemia in the multiple endocrine neoplasia syndromes.

Multiple endocrine neoplasia includes disorders with hyperfunction of two or more endocrine tissues. In MEN type 1, hyperfunction of the parathyroid glands causing hypercalcemia is the most common clinical presentation. In vitro, suppression of parathyroid tissue by calcium is similar, but the set-point of hyperplastic tissue is shifted as compared with normal. The gene for MEN-1 has been localized to chromosome 11 and is linked to the basic fibroblast growth factor gene. Parathyroidectomy results in a high failure rate with recurrent hyperparathyroidism or autonomous graft function in autotransplanted tissue. Family screening is recommended once every 5 years in first-degree relatives. The approach to hyperparathyroidism in MEN-2 (2A) must be individualized during surgery for medullary thyroid carcinoma. Hyperparathyroidism in MEN-3 (2B) is often associated with normal serum calcium and may not require intervention.

Chromogranin A↗