[New in vitro analysis tested: bleeding time is still the best method for evaluation of primary hemostasis]. / Ny in vitro-analys prövad: blödningstid fortfarande bästa metod för test av den primära hemostasen.

The need is great for a simple, cheap and readily accessible method for the evaluation of primary hemostasis in work-ups at both out-patient clinics and units caring for surgical or intensive care patients. PFA-100 is a recently introduced instrument for in vitro testing of platelet function. We report experiences from Stockholm, Gothenburg and Malmo of PFA-100 measurements performed on samples from healthy controls and from patients with von Willebrand disease or platelet disorders. It is shown that the PFA-100 system has a high sensitivity for von Willebrands disease, while the sensitivity for hereditary platelet dysfunction is low. In its present design this new device could not replace the template bleeding time as a screening test for primary hemostasis.

Differentiation of human parathyroid cells in culture.

Continuous culture of parathyroid cells has proven difficult, regardless from which species the cells are derived. In the present study, we have used a defined serum-free low calcium containing medium to culture human parathyroid cells obtained from patients with parathyroid adenomas due to primary hyperparathyroidism. No fibroblast overgrowth occurred, and the human parathyroid chief cells proliferated until confluent. After the first passage the cells ceased to proliferate, but still retained their functional capacity up to 60 days, demonstrated by Ca(2+)-sensitive changes in the release of parathyroid hormone (PTH) and as adequate cytoplasmic calcium ([Ca2+](i)) responses to changes in ambient calcium as measured by microfluorimetry. Low calcium concentrations enhanced, and vitamin D(3) and retinoic acids (RA) dose-dependently inhibited cell proliferation during the first passage, as determined by [(3)H]thymidine incorporation, immunohistochemistry for proliferating cell nuclear antigen and cell counting. Signs of differentiation were present as the set-points, defined as the external calcium concentration at which half-maximal stimulation of [Ca2+](i) (set-point(c)), or half-maximal inhibition of PTH release (set-point(p)) occur, were higher in not proliferating compared with proliferating cells in P0. Inhibition of cell proliferation was accompanied by signs of left-shifted set-points, indicating a link between proliferation and differentiation. The results demonstrate that human parathyroid chief cells cultured in a defined serum-free medium can be kept viable for a considerable time, and that signs of differentiation occur after proliferation has ceased. The low calcium stimulated cell proliferation may also be inhibited by vitamin D and RA.

Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor.

Familial hyperparathyroidism (HPT), characterized by hypercalcemia and hypercalciuria, and familial benign hypocalciuric hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. The calcium-sensing receptor (CaR) regulates PTH secretion and renal calcium excretion. Heterozygous inactivating mutations of the gene cause FHH, whereas CaR gene mutations have not been demonstrated in HPT. In a kindred with 20 affected individuals, the hypercalcemic disorder segregated with inappropriately higher serum PTH and magnesium levels and urinary calcium levels than in unaffected members. Subtotal parathyroidectomy revealed parathyroid gland hyperplasia/adenoma and corrected the biochemical signs of the disorder in seven of nine individuals. Linkage analysis mapped the condition to markers flanking the CaR gene on chromosome 3q. Sequence analysis revealed a mutation changing phenylalanine to leucine at codon 881 of the CaR gene, representing the first identified point mutation located within the cytoplasmic tail of the CaR. A construct of the mutant receptor (F881L) was expressed in human embryonic kidney cells (HEK 293), and demonstrated a right-shifted dose-response relationship between the extracellular and intracellular calcium concentrations. The hypercalcemic disorder of the present family is caused by an inactivating point mutation in the cytoplasmic tail of the CaR and displays clinical characteristics atypical of FHH and primary HPT.

Regulation of human eosinophil migration across lung epithelial monolayers by distinct calcium signaling mechanisms in the two cell types.

In asthmatic patients, eosinophils massively infiltrate the lung tissues and migrate through lung epithelium into the airways. The regulatory mechanisms involved are obscure. We studied the role of calcium in the migration of human eosinophils across monolayers of human lung epithelial H292 cell line cells induced by combined chemotactic solutions of platelet-activating factor and C5a. The transepithelial migration of eosinophils was attenuated by depletion of the external Ca2+ in the migration system, whereas the eosinophil migration itself was unaffected as evidenced by measuring eosinophil chemotaxis in the Boyden chamber in the absence of epithelial cells. Buffering of intracellular Ca2+ in eosinophils with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibited both eosinophil transepithelial migration and eosinophil chemotaxis in the Boyden chamber, suggesting the importance of intracellular Ca2+ in eosinophil transmigration. Although loading of BAPTA/AM or addition of thapsigargin to the epithelial cells effectively changed their cytoplasmic free Ca2+ concentrations, neither of these treatments affected transepithelial migration of eosinophils. Interestingly, addition of La3+ (0.2 mM) to epithelial cells suppressed eosinophil transmigration whereas addition of La3+ to eosinophils did not. Taken together, these results show the importance of Ca2+ in eosinophil migration across lung epithelium and support a distinctive regulatory role of intracellular and extracellular Ca2+ for the two cell types involved in this process; i.e., the transmigration of human eosinophils across a monolayer of lung epithelial cells is regulated by the intracellular Ca2+ in eosinophils, whereas the ability of the lung epithelial cell monolayer to allow eosinophil passage is dependent on the extracellular Ca2+.

Interleukin-6 induced suppression of bovine parathyroid hormone secretion.

Calcium disturbances in the critically ill coincide with elevations of proinflammatory cytokines. The effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) on parathyroid hormone (PTH) secretion were investigated. IL-6 and TNF-alpha had no acute effect on PTH secretion in extracellular Ca2+ concentrations of 0.5, 1.25 and 3.0 mM. In contrast to TNF-alpha, cultures for 24 h in the presence of 10 ng/mL of IL-6 showed decreased PTH secretion by 51% and 29% in 0.5 mM and 1.25 mM Ca2+ respectively. Neither IL-6 nor TNF-alpha, affected cytoplasmic Ca2+ of the cells. We conclude that PTH secretion in vitro can be suppressed by IL-6 at clinically relevant concentrations. This suppression may aggravate hypocalcemia of the critically ill and attenuate the conventionally strong stimulation of the PTH release by reduction in serum calcium.

Endothelin plays an important role in the endothelium-dependent vasodilatation in the human forearm.

UNLABELLED: Endothelium-dependent vasodilatation (EDV) in humans has been evaluated mainly by local infusion of a muscarinic-receptor agonists in the forearm. It has been postulated that the function of the vasodilator nitric oxide (NO) can be evaluated with this technique. However, the role of the vasoconstrictor endothelin in this model has not been investigated. METHODS: Ten male hypertensive and seven male normotensive subjects were subjected to measurements of forearm blood flow (FBF) by venous occlusion plethysmography during local intra-arterial infusion of metacholine (4 microg/min) or nitroprusside (10 microg/min). In parallel, forearm venous plasma endothelin (ir-ET) was determined. RESULTS: Metacholine and nitroprusside increased FBF 2.3 and 2.2 times the baseline level (6.6+/-2.8 SD ml/min/100 ml tissue) in hypertensive subjects and 5.1 times the baseline level (2.7+/-3.0 ml/min/100 ml tissue) for both drugs in the normotensive subjects. None of the drugs induced any significant changes in ir-ET levels in any of the groups (baseline 1.5+/-0.4 pmol/l in hypertensive and 1.1+/-1.2 pmol/l in normotensive subjects). However, in the hypertensive subjects, the individual change in venous ir-ET levels during infusion with metacholine, but not with nitroprusside, was inversely related to the degree of vasodilatation induced by this agent (r = -0.71, p < 0.02). A similar correlation coefficient (r=-0.69) was found in healthy subjects. CONCLUSION: Muscarinic-receptor-agonist-stimulated vasodilatation in the human forearm, thought mainly to reflect NO synthesis, was inversely related to the change in endothelin levels, suggesting an important role for this endothelium-derived vasoconstrictor in this model of EDV.

Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation.

Osteolysis or osteosclerosis often occurs in bone tissue adjacent to chronic inflammatory processes. Numerous cytokines and inflammatory mediators have been implicated as osteoclast-activating agents, explaining inflammation-induced bone resorption. In many cases, the cause of the sclerosis seen in these lesions is less thoroughly investigated. We have studied the effects of thrombin and bradykinin, 2 inflammatory mediators, on the rate of proliferation in isolated human osteoblasts (hOBs). Thrombin, at and above 1 U/mL, stimulated the rate of thymidine incorporation into hOBs. The absolute cell number also increased, as measured by an assay based on the detection of cell metabolism. A synthetic peptide ligand for the thrombin receptor enhanced the rate of [3H]thymidine incorporation in hOBs, indicating that thrombin-induced proliferation is mediated via the tetheric thrombin receptor. The thrombin-induced proliferation was not affected by indomethacin, excluding prostanoids as mediators of this effect. Bradykinin did not affect either the rate of thymidine incorporation, or number of cells in long-term cultures of hOBs. In conclusion, the inflammatory mediator, thrombin, stimulates proliferation in isolated human osteoblasts probably via the recently described G-protein-coupled tetheric thrombin receptor. Thrombin may therefore be involved as a mediator of inflammation-induced sclerosis and bone formation.

Tyr1009 and Tyr1021 in the platelet-derived growth factor beta-receptor mediate agonist triggered calcium signalling.

Calcium signalling was studied in porcine aortic endothelial cells stably transfected with wild type or mutants of the human platelet-derived growth factor (PDGF) beta-receptor and fibroblast growth factor (FGF) receptor-1 (FGFR1). Phospholipase C-gamma (PLC-gamma) has a consensus binding site at phosphorylated Tyr1021 in the PDGF beta-receptor. The phosphorylated tyrosine at 1009 is a binding site for Syp/PTP1D, an adaptor molecule mediating Grb2/RAS signalling. Also, Tyr1009 has been shown to be a minor binding site for PLC-gamma; however previous data have indicated that it does not have any functional significance in PLC-gamma signalling. The concentration of cytoplasmic calcium ([Ca2+]i) was measured by microfluorometry and digital imaging. About 72% of the cells transfected with wild type PDGF beta-receptor responded to a challenge with PDGF-BB. Mutants in which both Tyr1009 and Tyr1021 in the PDGF beta-receptor were exchanged for phenylalanine totally lacked [Ca2+]i responses. However, in those with a single mutation at Tyr1009 or Tyr1021, 36% and 12% of the cells responded, respectively. In cells transfected with FGFR1 or FGFchim, with the kinase insert of FGFR1 replaced by the insert of the PDGF beta-receptor, a [Ca2+]i increases was observed in similar proportions of cells. The amplitudes of the growth factor-induced [Ca2+]i responses was comparable in the different transfectants. Thrombin, activating a G-protein coupled receptor, triggered [Ca2+]i peaks more rapidly, and in a higher proportion of cells compared to the growth factors. The present data indicate that both Tyr1009 and Tyr1021 alone and in cooperation mediate PDGF-BB triggered calcium signalling.

Vitamin D receptor gene polymorphism and parathyroid calcium sensor protein (CAS/gp330) expression in primary hyperparathyroidism.

Calcitriol, via its receptor (VDR), inhibits parathyroid hormone (PTH) secretion and cell proliferation. Physically linked polymorphic VDR alleles denoted b,a, and T, comprise a novel risk factor for postmenopausal primary hyperparathyroidism (pHPT) by their presumed coupling to reduced VDR expression. This study examines VDR gene polymorphisms, parathyroid calcium-regulated cytoplasmic calcium concentrations ([Ca2+]i) and parathyroid expression of a calcium sensor protein (CAS/gp330). Genomic DNA was obtained from 66 postmenopausal women with pHPT and 66 age-matched female controls. Polymorphic VDR alleles were detected after polymerase chain reaction (PCR) and restriction digestion. Cryosections of pathologic parathyroid glands from 41 of the patients were immunostained with a monoclonal anti-CAS/gp330 antibody. Homozygosity for the VDR alleles b, a, and T was overrepresented in pHPT (p < 0.01-0.05) but did not couple to ED50 for calcium-regulated [Ca2+]i. The enlarged parathyroid glands possessed heterogeneous down-regulation of CAS/gp330. This down-regulation was the least conspicuous in the BBgenotype, and these few patients generally had larger parathyroid lesions (p < 0. 05). The VDR haplotype baTis a risk factor for pHPT possibly by hampering the regulatory actions of calcitriol. In contrast the BAt haplotype seems to be underrepresented in pHPT and to couple to larger parathyroid lesions as well as less deranged CAS/gp330 expression and parathyroid cell function. HPT in these individuals may relate to genetic events principally altering the regulation of cell proliferation, rather than calcium sensing of the parathyroid cells.

Regulation of gp330/megalin expression by vitamins A and D.

BACKGROUND: A membrane-bound 550-kD Ca2+-binding glycoprotein belonging to the low-density lipoprotein (LDL) receptor superfamily has recently been identified as a putative calcium-sensing molecule. This molecule, known as gp330/megalin, is among several tissues present in the proximal tubule, parathyroid and placental cytotrophoblasts, in which a Ca2+-sensing function has been demonstrated. METHODS: Regulation of mRNA and protein expression of gp330/megalin were studied in a recently established cell line derived from rat kidney proximal tubule cells (IRPTCs), in human JEG-3 cells and in the mouse embryonal carcinoma cell line F9. RESULTS: In IRPTCs, quantification of mRNA and protein expression demonstrated two- to five-fold increases after addition of 10(-6) mol L(-1) all-trans-retinoic acid, 9-cis-retinoic acid or 1,25-dihydroxyvitamin D3, alone or in combination. Similarly, an increase in gp330/megalin mRNA expression was seen in JEG-3 cells cultured with vitamin D and retinoids, as well as when F9 cells were differentiated by incubation with retinoic acid and cAMP. The IRPTCs were immortalized by viral infection with the SV40 genome preceded by a temperature-sensitive promoter. Thus, by culture of the cells at 41 degrees C, SV40 genome transcription is inhibited and the IRPTC phenotype is reversed towards non-infected proximal tubule cells. At 41 degrees C, gp330/megalin mRNA expression was significantly increased compared with cells incubated at 34 degrees C. CONCLUSION: The results indicate a correlation between exposure to retinoic acid or vitamin D or induction of cell differentiation (by retinoic acid/cAMP in F9 cells or inhibition of SV40 transcription in IRPTCs) and an increase in gp330/megalin protein and mRNA expression.

Vitamin D receptor polymorphisms correlate to parathyroid cell function in primary hyperparathyroidism.

Calcitriol acts via its receptor (VDR) and inhibits PTH secretion and parathyroid cell proliferation. Increased prevalence of the polymorphic VDR alleles b, a, and T has been demonstrated in sporadic primary hyperparathyroidism. Sixty-two patients with primary hyperparathyroidism due to parathyroid adenoma (mean age, 69.5 +/- 1.4 yr) were genotyped for these VDR polymorphisms. Dispersed cells of the adenomas were exposed to increasing concentrations of extracellular Ca2+ and analyzed for PTH release and cytoplasmic Ca2+ concentrations. Ca2+-mediated PTH inhibition exhibited higher ED50 and less suppression in the cells of patients who were homozygous for the b, a, and T alleles (P < 0.05-0.10). When analyzing haplotypes, the patients with baT demonstrated a ED50 of 1.81 +/- 0.15 vs. 1.29 +/- 0.10 for BAt (P < 0.05). As VDR alleles were unrelated to parathyroid intracellular Ca2+, influences of polymorphic VDR alleles on PTH secretion seem to involve mechanisms other than the Ca2+-sensing protein of the parathyroid cell surface.

Propofol induces a lowering of free cytosolic calcium in myocardial cells.

BACKGROUND: The intravenous anaesthetic drug propofol has been shown to depress myocardial contractility. Ketamine, on the other hand, is a well-documented cardiovascular stimulant. These differences could possibly be due to different effects of the drugs on the calcium homeostasis of the myocardium. METHODS: The fluorescent intracellular probe fura-2 acetoxymethyl ester (fura-2/AM) was used in this in vitro investigation to study the influence of intravenous anaesthetic drugs on free cytosolic calcium concentration in suspensions of isolated rat myocardial cells. RESULTS: Addition of 0.5-2.0 micrograms/mL propofol resulted in a significant and dose-dependent decrease of free cytosolic calcium concentration in the myocardial cells, while addition of 0.25-2.5 micrograms/mL ketamine did not affect this concentration significantly. CONCLUSION: The results imply that the previously demonstrated negative inotropic effect of propofol could possibly be related to its influence on calcium availability in the myocardium.

Endothelin-induced calcium signaling and secretion in chief cells and fibroblasts from pathological human parathyroid glands.

Endothelins (ETs) are 21 amino acid peptides with vasoactive and mitogenic properties. The three isopeptides (ET-1, -2, and -3) and their receptors (E1A and ETB subtypes) display expression in numerous tissues and possibly mediate autocrine/paracrine actions. The present investigation shows that ET-1 triggers biphasic increases of the concentration of cytoplasmic Ca2+ ([Ca2+]i) in pathological human parathyroid cells. Both the peak and sustained [Ca2+]i increase, as well as the proportion of responding cells, are dose-dependent in the 10(-10)-10(-7) mol/L range of ET-1. In absence of external Ca2+, the ET-1-induced [Ca2+]i peak is attenuated. ET-3 has no effect on [Ca2+]i indicating functional dominance of the ETA receptor subtype. ET-1 (10 nmol/L) lowers parathyroid hormone secretion in 0.5 mmol/L but not in higher external Ca2+ concentrations, and parathyroid cell ET release is inhibited by increases of external Ca2+. Fibroblasts overgrowing the parathyroid chief cells during monolayer culture respond to ET-1 with biphasic [Ca2+]i increases or repetitive [Ca2+]i spikes, but show no response to elevation of external Ca2+. These findings imply that ET secretion and ET receptor expression may constitute an autocrine/paracrine mechanism in the regulation of human PTH secretion.

Expression and function of a CD4-like molecule in parathyroid tissue.

BACKGROUND: Parathyroid tissue expresses the T-lymphocyte antigens CD3 and CD4, and parathyroid CD3 has earlier been proposed to interact in the regulation of parathyroid hormone (PTH) release. METHODS: Anti-Leu3a, a monoclonal antibody recognizing CD4, was used to stain parathyroid tissue immunohistochemically, to influence PTH secretion from enzymatically dispersed parathyroid cells, and to immunoprecipitate parathyroid CD4. Northern blot and polymerase chain reaction were used to clarify the similarity between parathyroid and lymphocytic CD4. Serum PTH level was measured with an immunoradiometric assay in healthy control subjects and individuals with human immunodeficiency virus type 1. RESULTS: The parenchyma of normal and abnormal parathyroid tissue displayed strikingly variable CD4 expression. Immunoprecipitation showed a 56 kd molecule, and Northern blot and polymerase chain reaction confirmed the similarity with lymphocyte CD4. Anti-Leu3a inhibited preferentially low calcium-stimulated secretion of PTH from dispersed parathyroid cells, without discernible influences on the cytoplasmic calcium concentration of these cells. Individuals with human immunodeficiency virus type 1 displayed significantly lower serum PTH levels than healthy control subjects. CONCLUSIONS: The results suggest that the human parathyroid chief cell expresses a CD4 moiety, which seems to interact in the PTH release in vitro and in vivo and which seems to use another second messenger system than the structurally similar T-cell equivalent.

Imaging of Ca(2+)-induced cytoplasmic Ca2+ responses in normal and pathological parathyroid cells.

Ca(2+)-induced changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) were studied in bovine and normal and pathological human parathyroid cells using digital image analysis of fura-2-loaded cells. When raising external Ca2+ from 0.5 to 3.0 mmol L-1, about 95% of all cells reacted rapidly and simultaneously with sustained elevation of [Ca2+]i. In approximately two out of three bovine parathyroid cells, normal human cells and cells from most patients with hyperparathyroidism (HPT) the sustained phase was preceded by an overshooting [Ca2+]i transient. The proportion of cells, with such a transient was decreased in cells from severe cases of uraemic parathyroid hyperplasia only. However, pathological human cells from adenomas and normal-sized glands associated with adenomas, as well as cells from primary and uraemic hyperplasias, had lower peak and sustained levels than normal human and bovine cells. The results indicate that both normal and pathological parathyroid cells exhibit heterogeneity in their [Ca2+]i responses to elevation of external Ca2+. The Ca(2+)-induced [Ca2+]i transients and the sustained elevations are attenuated in pathological human parathyroid cells. However, the presence of the overshooting transient represents physiological variability rather than being a consequence of the pathophysiology associated with HPT.

Somatostatin and octreotide interfere with calcium signalling in pancreatic beta-cells but not in normal and pathological parathyroid cells.

Somatostatin and its analogue octreotide exert antisecretory actions in several endocrine tissues. Somatostatin and octreotide (100 nM) promptly and reversibly counteracted glucose stimulated elevations of the concentration of cytoplasmic Ca2+ ([Ca2+]i) in mouse pancreatic beta-cells. In contrast both somatostatin and octreotide failed to affect basal or high Ca2+ stimulated [Ca2+]i of normal bovine, and adenomatous or hyperplastic human parathyroid cells. Furthermore, PTH release of these cells was unaltered by 1-1000 nM somatostatin and octreotide. The results indicate that somatostatin and octreotide lack direct actions on parathyroid cells, which contrasts to their effects on calcium signalling in cells from pancreatic islets.

Effect of tris buffer on free cytosolic calcium in myocardial cells.

OBJECTIVE: To study the effect of tris buffer on free cytosolic calcium in vitro. DESIGN: Open, randomized, control trial of dispersed rat myocardial cells. SETTING: Experimental laboratory in a large university hospital. SUBJECTS: Dispersed myocardial cells from Sprague-Dawley rats. INTERVENTIONS: The influences of pure trometamol (tris) and a tris butter mixture, as well as conventional sodium bicarbonate on free cytosolic calcium in suspended rat myocardial cells were studied with the fluorescent intracellular probe fura-2. MEASUREMENTS AND MAIN RESULTS: Addition of pure trometamol (tris) resulted in a significant increase of free cytosolic calcium in myocardial cells suspended in a buffer containing 1.25 mM of ionized calcium. The actions of trometamol display a dose-dependency in relation to the concentration of external ionized calcium since the ionized calcium response was reduced in a buffer with 0.5 mM of extracellular ionized calcium. Furthermore, removal of external ionized calcium totally prevented trometamol induced increases of ionized calcium, indicating that this increase is dependent on transmembrane ionized calcium fluxes. When tris buffer mixture was investigated in 1.25 mM of calcium, as well as 0.5 mM of external ionized calcium, a decrease of ionized calcium was noted initially, followed by an increase during the observation period. Addition of sodium bicarbonate to the two experimental settings resulted in a more prominent initial decrease of ionized calcium, followed by a slower increase which did not reach the initial values during the 20-min observation period. Extracellular pH was also included as a variable. When the cells were suspended in a buffer containing 1.25 mM of ionized calcium with a pH of 6.80 instead of 7.40 (as above), addition of pure trometamol also resulted in an increase of ionized calcium; however, after 20 mins this increase was smaller as compared with the results above. When tris buffer mixture as well as sodium bicarbonate was added, initial decreases of ionized calcium were recorded, followed by smaller increases during the observation period, compared with the increase in buffers with a pH of 7.40. CONCLUSIONS: Pure trometamol (tris) induces an increase in free cytosolic calcium in suspended myocardial cells.

Parathyroid tissue in normocalcemic and hypercalcemic primary hyperparathyroidism recruited by health screening.

Parathyroid tissue from 57 women (mean age 65.5 years) with sporadic primary hyperparathyroidism (HPT) was analyzed mainly to clarify its characteristics versus tissue from those with normocalcemia. Patients were recruited by population-based health screening of menopausal women. Analysis of three or four total serum calcium values showed normocalcemia in 16 patients (mean 2.53 mmol/L); 20 and 21 of the women were consistently (mean 2.82 mmol/L) or intermittently (mean 2.59 mmol/L) hypercalcemic, respectively. Parathyroid operation demonstrated a single adenoma in 81% of the individuals, and these lesions were most prevalent and commonly dominated by oxyphil parathyroid cells in the persistently hypercalcemic patients. Chief cell hyperplasia (two or three abnormal glands) of the nodular type was found more often in the normocalcemic patients. Total glandular weight was the smallest (mean 270 mg) among the normocalcemic women and contributed to delicate decisions with regard to the extent of resection. Immunostaining of cryosections with a monoclonal antibody recognizing a putative Ca2+ sensor demonstrated variably heterogeneous down-regulation of the recognized glycoprotein in the pathologic parathyroid glands from all the individuals. Dose-response relations for PTH release and the cytoplasmic Ca2+ concentration ([Ca2+]i) were determined in Ca2+ 0.5-3.0 mmol/L by examining dispersed cells with radioimmunoassay and microfluorometry after fura-2 loading, respectively. ED50 for PTH release and [Ca2+]i and the [Ca2+]i concentrations at Ca2+ 3.0 mmol/L were the least deranged in cells from pathologic glands of the normocalcemic patients. The findings substantiate that the abnormal parathyroid tissue of normocalcemic HPT principally is characterized by the same, albeit less extensive, morphologic and functional derangements, which consistently have been demonstrated in patients with HPT accompanied by hypercalcemia and detected clinically.

Modulation of calcium signalling and proliferation in monoblastoid U-937 cells.

The effects of TPA (12-0-tetradecanoylphorbol-13-acetate) and G-protein modulators on the concentration of cytoplasmic Ca2+ ([Ca2+]i), cytoplasmic pH and cell growth were investigated in monoblastoid U-937 cells. The G-protein activator NaF causes a dose-dependent increase of [Ca2+]i, that is partially sensitive to inhibition by pertussis toxin. The [Ca2+]i rise appears to come mainly from extracellular sources, and the Ca2+ influx is mediated by channels insensitive to the Ca2+ blocker verapamil. The Ca2+ ionophore ionomycin causes a biphasic rise of [Ca2+]i, reaching steady state levels slightly higher than those attained with NaF. TPA per se has no effect on [Ca2+]i, but potently reverses the NaF or ionomycin induced [Ca2+]i rise. Also, TPA partially counteracted the acidification induced by NaF. Both NaF and ionomycin per se had no effect on cell growth but partially counteracted TPA induced growth inhibition. Interferon-gamma and tumor necrosis factor-alpha did not affect [Ca2+]i by themselves but lowered the [Ca2+]i of NaF stimulated cells. The cytokines had no effect on cytoplasmic pH. This study indicates that elevations of [Ca2+]i in themselves does not trigger proliferation, but alterations of [Ca2+]i modulates the regulation of U937-cell growth.

Effects of calcium channel modulators on the regulation of cytoplasmic Ca2+ and hormone secretion of parathyroid cells.

Effects of Ca(2+)-channel modulators were examined in human, bovine and murine parathyroid tissue. In 0.5 mM external Ca2+, 100 microM verapamil inhibited parathyroid hormone release, stimulated uptake and efflux of 45Ca and raised cytoplasmic Ca2+ concentration ([Ca2+]i). However, in 3.0 mM Ca2+ the drug only affected efflux. Methoxyverapamil (50 microM) inhibited parathyroid hormone release in 0.5 mM but stimulated secretion in 3.0 mM Ca2+. BAY K 8644 (10 microM) had similar actions as verapamil on parathyroid hormone release and [Ca2+]i, whereas nifedipine (1 microM) and diltiazem (50-100 microM) lacked effects. Despite the lack of voltage-dependent Ca2+ channels in parathyroid cells, drugs with established actions on such channels affect [Ca2+]i and parathyroid hormone release. However, these actions are not sufficiently pronounced and tissue specific to allow their use for correcting hyperparathyroidism.