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.

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.

Calcium sensing by human medullary thyroid carcinoma cells.

Regulation of the cytoplasmic calcium concentration ([Ca2+]i) was studied in fura-2-loaded C-cells from two human medullary thyroid carcinomas (MTC). K+ depolarization induced sustained rise of [Ca2+]i reversed by verapamil. Elevation of external Ca2+ from 0.5 to 3.0 mM triggered regular oscillations or steady-state increases of [Ca2+]i. In Ca(2+)-deficient medium Sr2+ caused steady-state increase or oscillations of the 340/380 nm fluorescence ratio. The Ca2+ and Sr2+ actions were partially reversible by verapamil. La3+ and Ce3+ elicited transient [Ca2+]i peaks independent of external Ca2+, but no oscillations. The results indicate that human MTC cells express a parathyroid-like Ca2+ sensor coupled to intracellular mobilization and influx of Ca2+. A voltage-dependent Ca2+ influx may be of importance for the oscillations of [Ca2+]i.

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.

Neomycin interacts with Ca2+ sensing of normal and adenomatous parathyroid cells.

Effects of the polyvalent cationic antibiotic neomycin on regulation of the cytoplasmic Ca2+ concentration ([Ca2+]i) were studied in normal and adenomatous human, and bovine parathyroid cells. Parathyroid hormone (PTH) release was also measured in the bovine cells. Elevation of extracellular Ca2+ from 0.5 to 3 mM caused biphasic increase of [Ca2+]i and inhibition of PTH release. In low external Ca2+ neomycin inhibited PTH release and virtually only triggered the [Ca2+]i transient. In contrast [Ca2+]i was lowered and PTH release stimulated by neomycin in the presence of 3.0 mM Ca2+ or 7 mM Mg2+. These actions of Ca2+ and neomycin on [Ca2+]i were qualitatively similar but less pronounced in the adenomatous than normal human parathyroid cells. Some effects of neomycin were thus similar to those induced by other cationic agents interacting with the Ca2+ receptor mechanism on the parathyroid cell surface, whereas others may involve phospholipase C inhibition, protein kinase C activation or a direct reduction of the Ca2+ influx.

Effects of the antihypercalcemic drugs gallium nitrate and pamidronate on hormone release of pathologic human parathyroid cells.

BACKGROUND: Gallium nitrate and the bisphosphonates pamidronate and its dimethylated derivative comprise antihypercalcemic drugs with actions on bone. This study examines the in vitro effects of these compounds on human parathyroid cells. METHODS: Parathyroid hormone (PTH) release and the concentration of cytoplasmic calcium ion (Ca2+) of dispersed cells from parathyroid glands of 27 patients with sporadic primary or uremic hyperparathyroidism was measured. RESULTS: In 1.25 mmol/L external Ca2+, 200 mumol/L gallium nitrate inhibited PTH release from preparations of primary and uremic hyperparathyroidism by 14% and 22%, respectively. Similar reductions were evident also in 0.5 and 3.0 mmol/L Ca2+. The gallium nitrate-induced suppression of PTH release was dose dependent in the 2 to 200 mumol/L range. Cytoplasmic Ca2+ concentration displayed a biphasic rise on elevation of external Ca2+ and remained unaffected by gallium nitrate. None of the bisphosphonates altered PTH release of pathologic human or normal bovine parathyroid cells. CONCLUSIONS: The results support clinical usefulness of gallium nitrate through its dual actions on bone and the parathyroid. The findings substantiate that gallium may reduce PTH release by stabilization of the plasma membrane rather than by interference with the surface cation receptor mediating Ca2+ regulation of the secretion.

Expression and function of a CD3-like molecule on normal and abnormal human parathyroid cells.

BACKGROUND: Normal and abnormal human parathyroid tissue express the T-lymphocyte protein CD4, and parathyroid and lymphocyte cells show similarities with respect to mechanisms of calcium permeability and regulation of the cytoplasmic calcium concentration. METHODS: Anti-Leu4, a monoclonal antibody recognizing the T-lymphocyte glycoprotein complex CD3, is used to immunohistochemically stain normal and abnormal human parathyroid cells and to explore influences on the parathyroid hormone (PTH) secretion of enzymatically dispersed parathyroid cells. RESULTS: Parathyroid glands of patients with different forms of hyperparathyroidism displayed variable expression of the anti-CD3 reactive complex. The stainings correlated both positively and inversely to immunoreactivity for a previously defined calcium sensor, the decreased expression of which may constitute a molecular basis for hyperparathyroidism. Incubation of parathyroid cells with the anti-Leu4 antibody inhibited PTH secretion and reduced its sensitivity to external calcium without influence on parathyroid cytoplasmic calcium concentration. CONCLUSIONS: The results suggest that the human parathyroid cells express a CD3-like molecule with the ability to interact in PTH release.

Long-term effects of parathyroid operation on serum calcium and parathyroid hormone values in sporadic primary hyperparathyroidism.

BACKGROUND: This study investigates 410 persons (median age, 67 years) who underwent parathyroid operation for sporadic primary hyperparathyroidism 6 to 32 years (mean, 14.2 years) before 1991. METHODS: Patient records and operative specimens were scrutinized, the patients answered a questionnaire, and fasting serum samples were analyzed for calcium, albumin, creatinine, and intact parathyroid hormone (PTH) values. RESULTS: After primary parathyroid operations were performed with a conservative surgical approach, persistent and recurrent hypercalcemia were noticed in 3.7% and 1.7% of patients, respectively, whereas 4.7% of patients required vitamin D substitution or had essentially mild hypocalcemia. The PTH values were generally increased in patients with postoperative hyperparathyroidism, low in those with vitamin D substitution, and normal to elevated in the patients with hypocalcemia and in those with postoperative normocalcemia. The mean serum creatinine concentration was just below the upper reference range and correlated strongly with serum PTH value. No significant differences in serum PTH values were present between the normocalcemic patients and matched control patients after operation (n = 107), but the patients who underwent operation exhibited greater variation in the PTH concentration. CONCLUSIONS: The results substantiate the efficacy of parathyroid operation in sporadic primary hyperparathyroidism. Biochemical derangements compatible with secondary hyperparathyroidism may evolve during long-term follow-up and contribute to decreases in serum calcium values and increases in serum PTH values of these patients.

Incidence, structure, and function of enlarged parathyroid glands discovered accidentally during thyroid surgery.

BACKGROUND: Operation on rare patients with mainly a severe renal stone disease and considerably elevated urinary calcium excretion has substantiated the association of parathyroid gland abnormalities with normocalcemia. This study examines incidence, structure, and functional characteristics of enlarged parathyroid glands of patients with normocalcemia scheduled for thyroid surgery. METHODS: Eleven enlarged parathyroid glands weighing 110 to 1000 mg were discovered in 9 (1.5%) of 594 patients with normocalcemia undergoing thyroid operation. The preoperative total serum calcium concentration was 2.30 to 2.52 mmol/L and less than 2.38 mmol/L in four of the nine patients. Intact serum parathyroid hormone and alkaline phosphatase levels were elevated in only one individual, and all patients showed normal serum creatinine values. RESULTS: All but three of the 11 enlarged parathyroid glands exhibited microscopic abnormality on routine histopathologic examination, including staining for cytoplasmic fat with oil red 0. Immunohistochemical staining with a monoclonal antibody recognizing the functionally important calcium receptor of the parathyroid cell surface and analysis of the calcium-regulated cytoplasmic Ca2+ concentration of dispersed parathyroid cells substantiated that only a single gland of 130 mg had no discernible functional abnormality. CONCLUSIONS: The findings underline the diagnostic difficulties of parathyroid histopathology and support the presence of disturbed parathyroid hormone secretion even in normocalcemic patients with enlarged parathyroid glands. The functional derangement of these glands substantiates the indication for their surgical excision even in patients exhibiting midnormal serum calcium concentrations, although their possible contribution to the development of a clinically overt hyperparathyroidism can only be speculated.

Hyperparathyroidism of multiple endocrine neoplasia type 1: candidate gene and parathyroid calcium sensing protein expression.

BACKGROUND: Hyperparathyroidism affects most patients with multiple endocrine neoplasia type 1 (MEN 1). This study investigates expression of the candidate MEN1 gene phospholipase C beta 3 (PLC beta 3) and expression and function of a putative calcium sensing protein (CAS) in hyperparathyroidism of MEN 1. METHODS: In 31 parathyroid glands from 17 patients with MEN 1, CAS distribution was studied immunohistochemically and parallel sections were explored for PLC beta 3 mRNA expression by in situ hybridization. Enzymatically dispersed parathyroid cells were analyzed for cytoplasmic calcium concentrations [Ca2+]i and parathyroid hormone (PTH) release. RESULTS: All glands exhibited a heterogeneously reduced CAS immunoreactivity, especially meager in nodularly assembled parathyroid cells. Calcium regulated [Ca2+]i and PTH release tended to be more deranged in the glands possessing the lowest immunostaining. Parathyroid PLC beta 3 invariably was homogeneously expressed, and this included even MEN 1 patients with reduced PLC beta 3 expression in endocrine pancreatic tumors. CONCLUSIONS: The findings support variable calcium insensitivity of [Ca2+]i and PTH release in hyperparathyroidism of MEN 1, apparently coupled to heterogeneously reduced CAS expression. For clarification of the role of PLC beta 3 in MEN 1 parathyroid tumorigenesis further study of this protein is required.

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.

Relationship between abnormal regulation of cytoplasmic calcium and elevated blood pressure in patients with primary hyperparathyroidism.

Primary hyperparathyroidism (HPT) is characterised by a defective calcium sensitivity of the parathyroid glands. HPT is, furthermore, associated with a high prevalence of hypertension. In the present study BP was measured before operation, during surgery and after operation in 42 HPT patients and in 15 control subjects operated for non-toxic goitre. Parathyroid tissue was removed from all patients and the concentration of cytoplasmic calcium [Ca2+]i was determined in vitro in dispersed single cells by means of microfluometry at extracellular calcium concentrations of 0.5 mM and 3.0 mM. The SBP levels were found to be raised both before (158 +/- 23 (SD) mmHg vs. 144 +/- 24 mmHg in controls, P < 0.05), during surgery (maximal level 167 +/- 22 mmHg vs. 146 +/- 16 mmHg in controls, P < 0.01) and after operation (maximal level 180 +/- 26 mmHg vs. 148 +/- 20 mmHg in controls, P < 0.001) in the HPT subject when compared with controls. SBP during surgery was found to be related to the in vitro measured [Ca2+]i in the parathyroid cells at 3.0 mM extracellular calcium concentration or to the ratio of [Ca2+]i at 3.0 mM-0.5 mM (r = -0.25 and -0.27, respectively; P < 0.05). The degree of suppression of PTH release in vitro at 3.0 mM extracellular calcium was found to be related to both systolic and diastolic BP (r = 0.57 and r = 0.53, respectively; P < 0.05) before surgery. In conclusion, BP was found to be raised in HPT patients both before operation as well as during surgery and after operation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Different secretory actions of pancreastatin in bovine and human parathyroid cells.

Chromogranin A is an acidic protein that is costored and cosecreted with parathyroid hormone (PTH) from parathyroid cells. Pancreastatin (PST), is derived from chromogranin A, and inhibits secretion from several endocrine/neuroendocrine tissues. Effects of different pancreastatin peptides were investigated on dispersed cells from bovine and human parathyroid glands. Bovine PST(1-47) and bovine PST(32-47) inhibited PTH release from bovine cells in a dose-dependent manner. The former peptide was more potent and suppressed the secretion at 1-100 nM. This inhibition was evident in 0.5 and 1.25 mM, but not in 3.0 mM external Ca2+. Both peptides failed to alter the concentration of cytoplasmic Ca2+ ([Ca2+]i) of bovine cells. Human PST(1-52) and PST(34-52) did not affect PTH release or [Ca2+]i of parathyroid cells from patients with hyperparathyroidism, nor [Ca2+]i of normal human parathyroid cells. Furthermore, bovine PST(1-47) and bovine PST(32-47) failed to alter the secretion of abnormal human parathyroid cells. The study indicates that PST exerts secretory inhibition on bovine but not human parathyroid cells, and that this action does not involve alterations of [Ca2+]i.

[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.

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.

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.

PDGF-BB triggered cytoplasmic calcium responses in cells with endogenous or stably transfected PDGF beta-receptors.

Platelet-derived growth factor-BB (PDGF-BB) triggered signal transduction was investigated in human foreskin fibroblasts with endogenous PDGF beta-receptors, and porcine aortic endothelial (PAE) cells with stably transfected PDGF beta-receptors. Immunoprecipitation and immunoblotting showed that PDGF induced dose-dependent autophosphorylation of PDGF beta-receptor, and the PLC-gamma associates with autophosphorylated PDGF beta-receptors and becomes phosphorylated. Activation of PLC-gamma is known to induce fluctuations of the concentration of cytoplasmic calcium ([Ca2+]i). Microfluorometry and digital imaging were employed for measurements of the concentration of [Ca2+]i. In both cell types the growth factor induced four types of [Ca2+]i responses; no rise, a small and sluggish monophasic rise, a biphasic rise with an initial transient peak followed by a sustain elevation, and finally regular oscillations. The frequencies and amplitudes of the oscillatory responses were independent of agonist concentration after stimulation with PDGF-BB. Latency, the period from application of stimulus to the first [Ca2+]i peak, was reduced at higher concentrations of agonist. Also, the proportion of responding cells increased with higher concentrations of ligand. Oscillations of [Ca2+]i were elicited at submaximal concentrations of agonist. In PAE cells PDGF-BB triggered a single [Ca2+]i peak in absence of external Ca2+. Ligand-induced oscillations and sustained increases of [Ca2+]i were counteracted by the inorganic Ca2+ channel blocker Ce3+. These results show that similar types of [Ca2+]i responses occur in different cell types independently of whether the PDGF beta-receptors are expressed endogeneously or after transfection. Potentially, the different [Ca2+]i responses have distinct physiological consequences.