Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.     

Blastic OKT6-positive proliferation preceding malignant histiocytosis

A 45-year old male presented latero-cervical lymphoadenopathy. Biopsy revealed a malignant proliferation of immature "lymphoid" cells bearing T6 antigen and HLA-DR but negative for other lymphoid markers, suggesting a phenotype similar to Langerhans cells. The patient did not receive any therapy and six months later developed a histologically typical malignant histiocytosis, involving spleen and liver. Other reported cases of lymphoid malignancies (two bearing the T6 antigen on blast cells) preceding malignant histiocytosis were found and compared with ours. Most of these cases were characterized by the pediatric age of the patients and were presented as acute leukemias exhibiting, in at least some of them, reliable T-cell markers. Our case appears to represent, on the other hand, a blastic proliferation of precursors of both histiocytes and Langerhans dendritic cells which eventually progressed to malignant histiocytosis. The relevance of this observation in the debate on the origin of Langerhans cells and the relationships existing between macrophages and dendritic cells is discussed.     

Na+/H+ exchange in Ehrlich ascites tumor cells. Regulation by extracellular ATP and 12-O-tetradecanoylphorbol 13-acetate

The effects of extracellular ATP and/or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the intracellular pH of Ehrlich ascites tumor cells were measured using both distribution of [14C]5,5-dimethyloxazolidine-2,4-dione, and the fluorescent indicator 5(6)-carboxyfluorescein. Micromolar concentrations of extracellular ATP induce a biphasic change in the intracellular pH characterized by a rapid acidification of 0.04 pH units followed by an alkalinization of 0.11 pH units. Concurrently with the alkalinization, an increase in the total cellular [Na+] from 37.5 to 45.0 mM is observed. The pH change is half-maximally activated by 0.5-2.5 microM extracellular ATP. The intracellular alkalinization, but not the initial acidification, phase requires extracellular Na+, with half-maximal alkalinization in the presence of 24-32 mM Na+, and is inhibited by amiloride. Exposure of Ehrlich ascites tumor cells to TPA alone produces a slight alkalinization of approximately 0.04 pH units. Conversely, preincubation of the cells with TPA partially inhibits the ATP-induced changes in intracellular pH. Under identical conditions TPA also inhibits the ATP-induced increase in the cytosolic [Ca2+]. The half-maximal dose for both effects is produced by 3-10 nM TPA. These data indicate that extracellular ATP triggers the activation of Na+/H+ exchange. Furthermore, activation of protein kinase C mediates at least part of the Na+/H+ exchange, although a second mechanism may also exist.     

Serotonin transport in isolated platelet granules. Coupling to the electrochemical proton gradient

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in intact 5-hydroxytryptamine (serotonin) containing dense granules. The granules were isolated and purified from other subcellular organelles under isotonic conditions utilizing a newly developed continuous density gradient of Percoll. As measured by [14C]methylamine distribution, isolated granules suspended in a highly buffered medium at pH 7.0 had an intragranular pH of 5.40, independent of the pH of the external medium. This pH gradient could be collapsed by the addition of 60 mM ammonia. In the presence of Mg-ATP, a transmembrane potential (delta psi) of 30-40 mV, inside positive, was generated and sustained for over 30 min, as measured by [14C]thiocyanate distribution. The addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a proton translocator, resulted in the reversal of the potential to negative values. The Mg-ATP-dependent generation of the delta psi was prevented by addition of dicyclohexylcarbodiimide and trimethyltin, inhibitors of proton-translocating ATPases in this and other subcellular organelles. Ammonia (1-50 mM) addition to highly buffered suspensions of serotonin granules caused a dose-dependent decrease in the delta pH, while thiocyanate added at varying concentrations produced a dose-related collapse of the delta psi and had no effect upon the delta pH. Both the delta pH and delta psi were found to independently drive accumulation of [14C]serotonin into the granules; stepwise collapse of each gradient resulted in a corresponding diminution of [14C]serotonin accumulation. The maximum rate and extent of [14C]serotonin uptake, however, were observed in the presence of both the delta pH and delta psi. The conclusions provide support for the existence of a proton-translocating ATPase in the serotonin granule membrane responsible for the generation of the delta pH and delta psi. Moreover, the results demonstrate a primary role for the electrochemical proton gradient (delta mu H+) in the carrier-mediated active transport of 5-hydroxytryptamine into the platelet granule.     

Kinematic study of the temporal coupling between the components of transport and manipulation during reaching and grasping movements

In this study the temporal coupling between transport and manipulation components of prehension movements was tested. For this purpose two experiments were carried out. In Experiment 1 six normal subjects were required to reach and grasp one of three spheres located at three different distances (Blocked trials). In Experiment 2 a visual perturbation paradigm was used in which the location of the object to be reached and grasped could change at onset of arm movement (Perturbed trials). The results of this study exclude a temporal coupling between events of transport and manipulation components. On the contrary they suggest that manipulation component organizes its time course having information about the time required to reach the object.     

Electrostatic control of the rate-determining step of the copper, zinc superoxide dismutase catalytic reaction

The dependence of the activity of bovine Cu,Zn superoxide dismutase on pH and ionic strength was extensively investigated in the ranges of pH 7.4-pH 12.3 and of ionic strength of 0.02-0.25 M. The results obtained indicate that two positively charged groups having pK values of approximately 10.1 and 10.8 are involved in the control of the activity. On the basis of previous work on the three-dimensional structure and on the chemically modified enzyme, these groups are likely to be lysine side chains, in particular Lys-120 and Lys-134. The oxidation state of the enzyme-bound copper ion at the steady state was found to be the same at either pH 7.4 or pH 11.5. The diffusion of superoxide ion into the active site, which is controlled by the positive charges around the active site itself, appears to be the rate-determining step of the dismutation reaction. NMR measurements of the relaxation rates of F- showed that this control also applies to the access of F- to the active site. Comparison of the nuclear relaxation rates of F- with the enzyme activity indicates that F- relaxation is controlled by the deprotonation of the group with pK approximately 10.8, which appears to be responsible for about 50% of the total activity measured at neutral pH.     

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [14C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.