Effect of parathyroid hormone on elastase release from human polymorphonuclear leucocytes

Acute and chronic renal failure are clinical states associated with secondary hyperparathyroidism and increased catabolism. It has been suggested that elevated proteolytic activity is present in the blood in these clinical states. It is, theoretically, possible that the excess blood levels of parathyroid hormone (PTH) in patients with these disorders stimulate release of proteases, since this latter process is calcium dependent and PTH enhances entry of calcium into cells. The present study examined the effect of PTH and its amino- and carboxyterminal fragments on elastase release from polymorphonuclear leucocytes (PMNL), and evaluated the mechanisms underlying such an action. 1-84 PTH stimulated elastase release from PMNL in a dose-dependent and time-dependent manner. This effect of the hormone was abolished by its inactivation as well as by the presence of EDTA. Verapamil, trifluoperazine and W-7 reduced but did not abolish the 1-84 PTH-induced stimulation of elastase release from PMNL. Phorbol ester (PMA) also stimulated elastase release but both PTH or PMA-induced elastase release was blunted by staurosporin, an inhibitor of protein kinase C. The 19-84 carboxyterminal PTH also produced significant stimulation of elastase release from PMNL but the amino-terminal 1-34 PTH or other peptide hormones (insulin, calcitonin, and ACTH) had no stimulatory effect on elastase release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation and inhibition of protein kinase C in cultured bovine parathyroid cells: effect on the release of C-terminal fragments of parathyroid hormone.

The response of the parathyroid gland to low Ca2+ may be mediated in part by protein kinase C (PKC). We assessed the effect of two PKC activators, SC-9 and SC-10, and one PKC inhibitor, H-7, on Ca(2+)-regulated PTH release and degradation in primary cultures of bovine parathyroid cells. Both SC-9 and SC-10 stimulated PTH release, compared to high Ca2+ alone, in parathyroid cells incubated in high Ca2+, with maximal PTH release of at least twofold occurring at a concentration of either activator of 10 nM (p less than 0.05). We have previously shown that another PKC activator, PMA, not only enhances PTH release in the presence of high Ca2+ but suppresses low Ca(2+)-stimulated PTH secretion. In the present study, neither SC-9 nor SC-10 caused a comparable suppression of PTH release at low Ca2+. However, the PKC inhibitor, H-7 (1 microM), blocked low Ca(2+)-stimulated (compared to the low Ca2+ control) PTH secretion by approximately 50% (p less than 0.01) and did not affect high Ca2+ suppression of PTH secretion. H-7 (1 microM) was able to oppose the stimulation of PTH release by the PKC activators SC-9, SC-10, and PMA at high Ca2+ and negated the PTH release-inhibiting effect of PMA at low Ca2+. Culture medium from these experiments was subjected to reversed-phase HPLC and the eluted fractions analyzed by RIA for the presence of intact and C-terminal fragments of PTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of proton release from cultured human myotubes to identify malignant hyperthermia susceptibility

BACKGROUND: Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. During general anesthesia, a life-threatening hypermetabolic state may occur resulting from increased release of Ca2+ from the sarcoplasmic reticulum in skeletal muscle. Diagnosis of MH susceptibility requires surgical muscle biopsies to measure force in response to chemical stimulation (in vitro contracture test, IVCT). Here, the authors investigated an alternative way of discriminating MH-susceptible (MHS) from normal (MHN) subjects by using cultured human myotubes and measuring proton release as an indicator of cellular metabolism. METHODS: Myotubes were stimulated with the Ca2+ release channel agonist 4-chloro-m-cresol (4-CmC), leading to metabolic activation and proton secretion. The rate of extracellular acidification was recorded with a silicon sensor chip. RESULTS: A stepwise increase in 4-CmC concentration led to a phasic-tonic increase in the acidification rate. The response, measured at different concentrations of 4-CmC, was considerably larger in cultures from MHS compared with MHN subjects and correlated well with the force response in the IVCT. CONCLUSIONS: The enhanced metabolism of cultured skeletal myotubes, likely originating from an increased myoplasmic Ca2+ concentration, can be monitored by studying the proton secretion rate. Because the method seems to be able to distinguish normal from pathologic phenotypes, it is a promising technique for possible future use in less invasive MH testing.     

Adrenal hemorrhage during the treatment of ulcerative colitis with adrenocorticotropic hormone.

Spontaneous adrenal hemorrhage is a rare condition that carries a high mortality. Its diagnosis may present considerable difficulty. Two instances of adrenal hemorrhage are reported, both occurring in patients with ulcerative colitis who had an acute exacerbation of their disease. Both patients were receiving adrenocorticotropic hormone therapy and it is thought that this was responsible for the hemmorrhage.     

Effect of human somatotropin hormone on cultured rat islets

Because growth hormone (GH) improves the insulin secretion capacity of isolated human fetal islets in vitro, we sought to show that it positively influences isolated rat islets. Islets isolated from Wistar albino rats by a modified automated system were cultured in media containing 87% RPMI 1640, 10% FCS, 2% antibiotic-antimycotic, and 1% L-glutamine for 12 +/- 2 days. The cultured islets were divided into two groups: growth hormone negative (Group I) and growth hormone positive (Group II). On the 5th day we observed a decrease in the islet cell counts in both groups (Group I 28% versus Group II 45%). On the 10th day, the decrease continued in the GH-negative group (59%), while the count remained stable in the GH-positive group. The viability of rat islets was determined by fluorescein diacetate (FDA) plus propidium iodide (PI) staining. In comparison to the peripheral green, central orange-red staining pattern of Group I islets upon fluorescent microscopy, Group II showed more compact islets. Cultured islets seemed to be brighter than those without GH in the cultured islets. In conclusion, we observed that 2 weeks of incubation in the presence of GH acts positively on cultured rat islets for both their amount and their viability.     

Secretion of high-molecular-weight adrenocorticotropic hormone from a pituitary adenoma in a patient without Cushing stigmata. Case report

The authors report a case in which a patient harbored a corticotroph macroadenoma that secreted biologically inactive high-molecular-weight adrenocorticotropic hormone (ACTH) as well as authentic ACTH 1-39. The secretion of the high-molecular-weight ACTH was determined using gel chromatography. The authors believe that these two molecules competed with each other at the ACTH receptor and, thus, the bioactivity of ACTH 1-39 was masked and Cushing features were not manifested in the patient. This type of silent corticotroph adenoma may be categorized as a clinically nonfunctioning adenoma. Plasmas from patients with silent corticotroph adenomas, which are identified by positive immunohistochemical staining of ACTH, should be frozen, stored, and analyzed using gel chromatography to examine whether the tumors produce and secrete high-molecular-weight ACTH.     

A case of adrenocorticotropic hormone (ACTH) deficiency

We report a case of ACTH deficiency. A 75-year-old man complained of anoxia, nausea and vomiting. Three years ago, he had an attack of loss of consciousness. On admission, his serum sodium level was down to 119.6 mEq.l-1. Plasma osmolality was low and urinary osmolality was high without edema, and he was diagnosed as having SIADH. After CRH test, rapid ACTH test and continuous ACTH test, he was diagnosed as having ACTH deficiency, and he was treated with steroids. One year later, he received urethrotomy due to urethrostenosis under spinal anesthesia with no trouble. In the next year, he was scheduled for sigmoidectomy due to sigmoid colon cancer under general anesthesia combined with epidural anesthesia. In the morning of his operation, he took hydrocortisone 10 mg per os. During operation, hydrocortisone 300 mg was given intravenously divided for three times. Plasma ACTH and aldosterone levels were below normal ranges, but serum cortisol was above the normal range. His operation was finished without troubles. Regarding this case, we discussed steroid therapy during anesthesia and operation.     

Surgical strategy in the management of non-small cell ectopic adrenocorticotropic hormone syndrome.

BACKGROUND. Non-small cell ectopic adrenocorticotropic hormone (ACTH) syndrome is a rare cause of hypercortisolism that may require surgery for either curative resection or palliative adrenalectomy. METHODS. We report our surgical experience with 41 patients with ectopic ACTH syndrome and no evidence of small cell lung cancer at initial evaluation. RESULTS. All 41 patients had documented hypercortisolism secondary to ectopic production of ACTH. Based on imaging study results, we determined that 21 patients had localized/resectable disease; eight patients had metastatic disease, and 12 patients had occult disease at examination. Of the 21 patients with localized disease, 16 (76%) were cured of ectopic ACTH by surgery (15 bronchial carcinoid, one pheochromocytoma). Patients with bronchial carcinoid had the greatest probability for cure of ectopic ACTH syndrome, and patients with thoracic primary tumor were more likely to be cured than patients with abdominal primaries. Of the eight patients who had metastatic disease, none were cured of the disease; five patients underwent bilateral adrenalectomy, and three patients were given medical therapy. Only one patient was alive after 5 years. Of the 12 patients who had occult disease, four patients were eventually cured of the disease (three bronchial carcinoid, one thymic carcinoid); one patient died of disease (small cell lung cancer), and seven patients still have occult disease. Nine of 12 patients with occult disease underwent bilateral adrenalectomy for surgical management of hypercortisolism. CONCLUSIONS. This study suggests that the most common primary focus of ectopic ACTH production is within the thorax with 25 of 34 (74%) identifiable tumors originating within either the thymus or bronchus. Adrenalectomy offers excellent palliation of hypercortisolism secondary to either occult or metastatic disease. Patients who initially have localized disease usually have bronchial carcinoids and have a high probability of cure with surgical resection (81%).     

Aprotinin increases release of von Willebrand factor in cultured human umbilical vein endothelial cells.

Through the perioperative administration of the proteinase inhibitor aprotinin, hemostasis can be improved and postoperative bleeding reduced after cardiac operations. The mechanism of action has been only partially clarified. The goal of our study was to investigate the influence of aprotinin on the synthesis of von Willebrand factor (vWF) in human endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultivated in vitro and incubated with different aprotinin concentrations (55, 100, and 215 mol/L). With all investigated aprotinin concentrations, there was an increase in vWF synthesis compared with basal secretion (p less than 0.001). When the HUVEC were preincubated with aprotinin and stimulated with thrombin, there was a further significant increase in vWF synthesis. HUVEC that, were first incubated with aprotinin and then stimulated with thrombin demonstrated a significant increase in vWF synthesis compared with basal secretion in nonincubated cells (p less than 0.0001). Also, compared with the cells that had received thrombin stimulation alone, the combination of aprotinin incubation and thrombin stimulation led to a significantly higher vWF concentration (p less than 0.05). Because vWF is necessary for the interaction with platelet factor glycoprotein Ib and platelet adhesion, the demonstrated increase in vWF synthesis could be one of the mechanisms of action of aprotinin leading to its blood-sparing effect.     

Antisense inhibition of vascular endothelial growth factor in human head and neck squamous cell carcinoma

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent paracrine angiogenic factor involved in angiogenesis. We determined whether antisense VEGF transfection can suppress angiogenic activity of a human squamous cell carcinoma of the head and neck (SCCHN) cell line. METHODS: Human SCCHN cell lines were screened for VEGF secretion by ELISA. The highest VEGF secreting cell line was transfected with an antisense VEGF vector. Endothelial cell migration assays were performed using the conditioned medium from the transfected clones. Tumorigenicity assays of the transfectants in nude mice were also performed. RESULTS: Antisense VEGF expression exhibited a 20-fold inhibition of VEGF secretion. The addition of conditioned medium from the antisense clones resulted in 50% reduction of endothelial migration. There was no effect on in vivo tumorigenicity. CONCLUSIONS: Antisense VEGF transfection effectively down-regulated VEGF secretion from SCCHN cells that had high VEGF secretion. Targeting VEGF expression may be useful for suppressing angiogenesis in head and neck cancer.     

Antisense oligonucleotides: inhibition of liver cell proliferation and in vivo disposition.

As oligomers inhibited hepatoma growth in vitro. These cells have oligomer-specific binding sites. The oligomers were taken up by liver and have potential for hepatic growth modulation in intact animals.     

Intraoperative measurement of adrenocorticotropic hormone in peripituitary blood in Cushing's disease

A new method of intraoperative measurement of adrenocorticotropic hormone for the localization of microadenomas in Cushing's disease is described and, in 21 cases, compared with preoperative bilateral sampling of the inferior petrosal sinus. By intraoperative measurement of adrenocorticotropic hormone in peripituitary venous blood, it was possible to determine a gradient for adrenocorticotropic hormone in 14 of 19 patients with surgically proven adenomas. In 4 of 6 patients in whom a false positive bilateral sample from the inferior petrosal sinus directed the surgeon to the wrong side of the pituitary, the localization could be correctly determined by intraoperative radioimmunoassay of adrenocorticotropic hormone.     

Ectopic adrenocorticotropic hormone syndrome caused by a carcinoid tumor of the thymus. A case report

Ectopic ACTH secretion accounts for less that 10% of all causes of endogenous Cushing's syndrome. Carcinoids are rare thymic tumors, and when associated with ACTH hypersecretion display local or distant aggressive behavior. A 32-year-old woman was admitted to the Endocrinology Unit for obesity, moon face, facial hirsutism, hyperpigmentation, and secondary amenorrhea. Laboratory test confirmed the hypercortisolism and excess ACTH, while dexamethasone suppressive test was negative. Thorax computed tomography (CT) showed an antero-superior mediastinal tumor invading the pericardium and left mediastinal pleura. A complete resection through median sternotomy of the tumor, pericardium and left mediastinal pleura was performed. After a one-year symptom-free period, hypercortisolism recurred, confirmed by laboratory findings. Although no signs of local recurrence were seen on thorax CT, left internal mammary lymph nodes involvement and vertebral body metastases at C7 and LI were found. Refractory electrolyte disturbances could not be corrected resulting in severe cardiac arrhythmia and death from cardiac arrest. The reported case draws attention on the aggressiveness of ACTH-secralso due to the refractory electrolyte disturbances with fatal outcome.     

Alloxan stimulation and inhibition of insulin release from isolated rat islets of Langerhans.

The rate of alloxan-induced insulin release was measured from rat islets maintained in a simple perifusion system. Insulin release during the five-minute exposure to alloxan reached its maximum rate after two to three minutes of the exposure and then rapidly declined. This insulin release was dependent upon extracellular calcium and was associated with an increased 45Ca uptake by isolated islets. Once exposed to alloxan, however, the islets did not release insulin when stimulated again with D-glucose or alloxan. These effects of alloxan on insulin release (stimulation and subsequent inhibition) and the increased 45Ca uptake were prevented by the presence of 3-0-methyl-D-glucose during the alloxan exposure. These findings indicate a close correlation between alloxan-induced insulin release and the subsequent inhibition of further insulin release. D-glucose, when present during the entire five-minute exposure to alloxan, protected competitively against alloxan inhibition of insulin release. In addition, D-glucose, when present immediately after brief (one to three minutes) alloxan exposures, reversed some of the subsequent inhibition of insulin release. These findings suggest that alloxan and D-glucose were competing for a common site on the beta-cell. The possibility of this site being a receptor responsible for the initiation of insulin release is discussed.     

The effect of phorbol ester on in vitro release of parathyroid hormone from abnormal human parathyroid cells

Intracellular events that regulate parathyroid hormone (PTH) release are not well understood. Cyclic AMP (cAMP) and cAMP-dependent protein kinases play a role in the regulation of release due to several agonists, but these factors do not fully explain PTH release that is mediated by extracellular ionized calcium (Ca++). A calcium-phospholipid-dependent (non-cAMP-dependent) protein kinase can be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether this protein kinase regulates PTH release, we examined the effect of TPA on PTH release from human parathyroid tissue. Cell suspensions of abnormal parathyroid tissue removed at surgery were prepared by enzymatic dispersion and incubated for several hours with and without 10(-7) mol/L TPA at low and high calcium levels. In ten preparations in the absence of TPA, increasing Ca++ from 0.25 to 2.5 mmol/L reduced PTH release to an average of 39% of maximal release (range, 11% to 67%). The effect on TPA on Ca++-regulated PTH release appeared biphasic. At low (0.25 mmol/L) Ca++ level, TPA suppressed PTH release to an average of 78% of maximal release without TPA (95% confidence interval, 67% to 88%) (p less than 0.01 compared to cells incubated without TPA). At high (2.25 mmol/L) Ca++ level, TPA augmented PTH release from an average of 39% of maximal release without TPA to 62% of maximal release without TPA (95% confidence level, 48% to 78%), an average augmentation of 22% (95% confidence level, 9% to 36%) (p less than 0.01 compared with cells incubated without TPA). TPA appeared to make PTH release independent of Ca++. Both inhibitory and stimulatory effects were dose dependent. Incubations with TPA demonstrated no toxicity as judged by trypan blue dye exclusion, linearity of PTH release, and cellular incorporation of tritiated leucine. TPA had no effect on the radioimmunoassay for PTH. We conclude that a calcium/phospholipid-dependent, non-cAMP-dependent protein kinase may play a role in mediating Ca++-regulated PTH release from abnormal human parathyroid cells. Its site of action and integration with other regulatory pathways remain to be determined.     

GUDC inhibits cytochrome c release from human cholangiocyte mitochondria.

Although ursodeoxycholic acid (UDC) is considered effective treatment for primary biliary cirrhosis (PBC), its mechanism of action is unclear. We tested the hypothesis that UDC is taken up by cholangiocytes and inhibits caspase 3-dependent apoptosis. We used the human cholangiocyte cell line (H69) and assessed it for expression and function of an apical sodium-dependent bile acid transporter (ASBT) by RT-PCR and uptake of tritiated taurocholic acid. We experimentally induced apoptosis in H69 cells using beauvericin (BV) and determined caspase 3 activation using a fluorogenic substrate and mitochondrial cytochrome c release (CC) into the cytosol by immunoblot analysis. We found that a functional ASBT is expressed by H69 cells as demonstrated by RT-PCR and bile acid uptake studies. Exposure of H69 cells to BV induced apoptosis in 39.4 +/- 1.3% of cells at 2 h (0.23 +/- 0.2% in controls). In contrast, when H69 cells were preincubated with GUDC (50 mM) for 24 h and then exposed to BV, apoptosis was inhibited by 23% (P < 0.03). In cholangiocytes pretreated with GUDC for 24 h and those treated with BV for 2 h, caspase 3-like activity was reduced by 79% and mitochondrial CC release was inhibited. In summary, the human cholangiocyte cell line H69 possesses a functional bile acid transporter, and GUDC decreases BV-induced apoptosis and inhibits activity of caspase 3 protease by blocking CC release from mitochondria. These preliminary results are consistent with our hypothesis that the beneficial effect of UDC on PBC may involve decreased apoptosis after GUDC uptake by cholangiocytes.