Secretion from bovine chromaffin cells acutely expressing exogenous proteins using a recombinant Semliki Forest virus containing an EGFP reporter.

Acute expression of recombinant proteins throughout a population of postmitotic bovine chromaffin cells was achieved using the Semliki Forest virus expression system (P. Liljestrom and H. Garoff (1991) Biotechnology 9:1356-1361). The virus was modified to express a green fluorescent protein, which faithfully reported the expression of the recombinant proteins. Two types of reporting virus were constructed: the first included a second subgenomic element, and the second an internal ribosome entry site. Both were used to express the recombinant proteins beta-galactosidase, 5HT3 receptor, or tetanus toxin light chain. Beta-galactosidase was used to quantify the rate of expression of recombinant protein in chromaffin cells, the 5HT3 receptor to trigger secretion, and the toxin to block secretion. The experiments clearly show that infection and expression of recombinant proteins throughout a population of chromaffin cells do not, per se, affect the rate and extent of triggered exocytosis, endocytosis, or membrane recycling pathways. The catecholamine content of the cell is unaltered, and the secretory mechanism can be accessed within a few hours after infection. This noncytopathic method of acutely expressing specific proteins at physiological levels in chromaffin cells offers a powerful new tool for dissecting the roles of many proteins implicated in exo- and endocytosis.

A novel use of differential equations to fit exponential functions to experimental data.

A procedure for fitting multi-exponential functions to experimental data is described. It is fast, requires no initial parameter estimates and is particularly suited to sums of several closely spaced exponentials. The method comprises the application of three well tried numerical techniques: (i) the signal is smoothed by representing it as an abbreviated Legendre series; (ii) the coefficients of a certain kind of differential equation are determined such that it's solution is the closest fit to the smoothed signal; and (iii) the amplitudes of the exponential components are determined, given the calculated values of the exponential rate constants. The method is computationally efficient, since determination of amplitudes and exponents involves the use of linear techniques, and therefore does not require multiple iterations, and the smoothed signal is contained in a handful of coefficients rather than as a lengthy time series. The severe ill-conditioning that is unavoidable in this problem is contained within the well-understood procedures of inverting a matrix and determining the roots of a polynomial. This method is particularly appropriate for analysis of data that may be modelled by a scheme of linked first-order reactions, describing for example the stochastic behaviour of ion channels, a chemical reaction, or the uptake and distribution of a drug within body compartments.

Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells.

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells. In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules. The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca2+ and MgATP. Although secretion requires Ca2+ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca2+ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides. These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca2+ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.

Growth factor expression during rat development: a comparison of TGF-beta 3, TGF-alpha, bFGF, PDGF and PDGF-R.

At least part of the mechanism underlying fetal development appears to be the production of a number of growth factors considered important in the process of tumour formation. Using immunocytochemistry, we have investigated the temporal and spatial pattern of expression of some of the important growth factors, by the fetus. We describe here the cellular localization of transforming growth factor beta 3 (TGF-beta 3), platelet derived growth factor (PDGF) and its receptor (PDGF-R), TGF-alpha and basic fibroblast growth factor (bFGF) in the fetal rat from day 13 to 21 of gestation. Using antisera raised against an N-terminal portion of TGF-beta 3, immunoreactivity peaked around day 16 and was seen predominantly within epithelial cells. However, using antisera raised against the C-terminal of this molecule immunoreactivity was seen exclusively within the extracellular matrix underlying adjacent epithelia, and was maintained up until day 21 of gestation. Strong expression of TGF-alpha was seen in cells of most organs throughout the gestation period studied. Immunoreactivity for bFGF, PDGF and PDGF-R peaked around day 18 in both epithelial and mesenchymal cells of all major organ systems and then declined by day 21. These data suggest distinct roles for each factor during embryogenesis and tumorigenesis.

A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.

Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed.

1. A general approach to the analysis of ensemble currents of ligand-gated channels is presented, with a variety of examples that include single and multiple agonist binding steps, desensitization and several blocking pathways. 2. The use of matrix methods to describe model reaction schemes leads to a simplification if the reaction scheme is irreversible: the product of the exponential relaxation rate constants is exactly equal to the product of the forward reaction steps. 3. This method of analysis applied to the bovine adrenal nicotinic receptor suggests that in the range of acetylcholine concentrations from 1 microM to 2 mM, a model with a single kinetically relevant agonist binding step is appropriate. 4. Complex models, to explain the presence of two desensitizing components in currents recorded from whole cells, may be discounted in favour of two distinct receptor types. 5. A simple model of open channel block is discounted, and desensitization of the blocked state proposed.

Parathyroid hormone-related peptide: expression in fetal and neonatal development.

Hypercalcaemia frequently complicates the clinical management of cancer. Many factors have been implicated in the pathogenesis of this humoral hypercalcaemia of malignancy, the most recent candidate being parathyroid hormone-related peptide (PTHrP). Until now, this peptide has been detected only in some normal and transformed adult tissues. In recent years, it has become apparent that tumours are capable of expressing and secreting factors previously elaborated only during fetal life. Many of these factors act to stimulate the growth of both tumour and fetal cells in an autocrine manner. The data presented here demonstrate that PTHrP is expressed in the human and rat fetus throughout gestation. Immunocytochemistry reveals a gestationally related, changing pattern of expression which is paralleled by changes in mRNA transcription. These data support the hypothesis that PTHrP may function as a fetal growth factor.

Parathyroid hormone-related peptide can regulate the growth of human lung cancer cells, and may form part of an autocrine TGF-alpha loop.

Parathyroid hormone-related peptide (PTHrP) and transforming growth factor-alpha (TGF-alpha) were found to stimulate proliferation of human lung cancer cells (BEN-57). TGF-alpha stimulated PTHrP secretion from these cells. The polyclonal antisera raised against PTHrP significantly inhibited the growth of BEN-57 cells, and also the proliferation induced by TGF-alpha. Treatment of cells for up to 10 days with either a PTHrP receptor antagonist (PTHrP(7-34)) or PTHrP antiserum significantly inhibited the subsequent growth of these cells. We suggest that PTHrP may be a component of a complex autocrine loop involving TGF-alpha.

The use of intracellular dialysis to study signal transduction coupling in the squid giant axon.

The squid giant axon has proved a useful model in the study of ionic channel gating, intracellular homeostasis and receptor-mediated signal transduction leading to generation of intracellular second messengers. In the latter category, previous studies on activation of adenylate or guanylate cyclase have used intact and intracellularly perfused axons to investigate the effects of extra- and intracellular agents on the transduction processes. However, the perfusion of the axon interior washes out many factors which may be important in the processes under study. We introduce here the use of porous cellulose dialysis tubing as a means to circumvent these problems. We find that this dialysis technique is a simple procedure to set-up, and the serotonin/G-protein/adenylate cyclase system can readily be studied in the dialysed axon. This approach should allow investigation under conditions which retain asymmetric transmembrane conditions.

The effect of botulinum toxin type D on the triggered and constitutive exocytosis/endocytosis cycles in cultures of bovine adrenal medullary cells.

The extracellular fluid phase marker, horseradish peroxidase, enters chromaffin cells when triggered to secrete catecholamine. This triggered uptake, like secretion, is abolished in cells pre-incubated with botulinum toxin. Endocytosis of horseradish peroxidase into unstimulated cells is unaffected by botulinum toxin but is inhibited when the temperature is reduced. Once internalised by the unstimulated cells, horseradish peroxidase is released back into the extracellular fluid, the rate of release being temperature sensitive but unaffected by carbamylcholine or botulinum toxin. These results suggest that triggered exocytosis is a necessary event to precede triggered endocytosis, and that botulinum toxin may affect only the triggered exocytosis/endocytosis cycle and not the constitutive cycle.

Membrane recapture and early triggered secretion from the newly formed endocytotic compartment in bovine chromaffin cells.

1. Recycling of secretory vesicles in cultured bovine adrenal medullary cells was investigated. 2. Extracellular horseradish peroxidase (HRP), a fluid phase marker, was taken up into cultured adrenal medullary cells following carbamylcholine-induced secretion of catecholamine. 3. The endocytosed HRP remained compartmentalized within the cell, migrating to a low density band on a Percoll density gradient. The endocytotic compartment was distinct from the major pool of catecholamine-containing chromaffin granules, which were found at much higher densities on the Percoll gradient. 4. The chromaffin granule membrane marker dopamine beta-hydroxylase was associated with the endocytosed HRP compartment as well as with the heavier chromaffin granules. 5. A subsequent challenge of the cells with carbamylcholine triggered the release of up to forty per cent of the endocytosed HRP. 6. The time course for secretion of the fluid phase marker was similar to that for catecholamine secretion. 7. Triggered release of HRP was dependent on extracellular calcium. The dependence on the extracellular calcium concentration was similar to that of catecholamine release. 8. Release of HRP could be triggered from electropermeabilized cells by raising the intracellular Ca2+ into the micromolar range. The intracellular Ca2+ dependence of triggered HRP release was similar to that for catecholamine release. 9. HRP could be secreted as early as 5 min, and as late as 2 h after endocytosis. 10. These data provide evidence that endocytotic vesicles can rapidly re-enter the secretory cycle. Endocytosed vesicles may therefore not have to recycle via the trans-Golgi reticulum to form high-density chromaffin granules in order to re-enter the regulated secretory pathway.

Parathyroid hormone-related peptide in normal human fetal development.

Parathyroid hormone-related peptide (PTHrP) has been detected in fetal serum and amniotic fluid. Using a combination of immunocytochemistry and molecular biology we have detected the peptide and its mRNA in a variety of fetal tissues throughout gestation. Tissue-specific mRNA isoforms were observed, the pattern of hybridization of which changed throughout gestation. In addition, the intensity and pattern of immunocytochemical localization of the peptide was found to vary over the time-period studied (8-30 weeks). PTHrP is expressed by a variety of tumours associated with the syndrome of humoral hypercalcaemia of malignancy and probably accounts for the hypercalcaemia by virtue of its limited amino acid homology with parathyroid hormone. These data demonstrate for the first time that PTHrP, a tumour-related peptide, is expressed during normal human fetal development, and suggest the possibility that it may function to regulate fetal calcium balance and growth in utero.

Parathyroid hormone related peptide can function as an autocrine growth factor in human renal cell carcinoma.

Parathyroid hormone related peptide (PTHrP) has been implicated in the cause of the hypercalcemia associated with a number of malignant tumours. The data presented here suggests that PTHrP (in addition to its known role of mediating hypercalcemia) may be involved in the autocrine regulation of growth of some tumours. Polyclonal PTHrP antiserum almost totally inhibited the growth of a human renal cell carcinoma cell line, known to secrete PTHrP, in vitro and growth was significantly inhibited by the competitive PTH antagonist PTH (3-34)NH2.

Parathyroid hormone-related peptide in the human fetal uro-genital tract.

Using a polyclonal antiserum raised against the first 34 amino acids of human parathyroid hormone-related peptide (PTHrP), we have localized PTHrP throughout the uro-genital tract of the human fetus aged between 8 and 40 weeks. Staining was present in the developing mesonephros, metanephros, gonads and in both the adrenal cortex and medulla. In particular, the developing mesonephric and metanephric renal tubules were intensely positive. Using Northern hybridization analysis we have detected a complex pattern of PTHrP mRNA transcripts ranging in size from 1.4 to 4.5 kb in early second trimester human fetal kidney. The presence of PTHrP in the mesonephros and metanephros provides evidence for a role for PTHrP in the regulation of fetal calcium metabolism. However, its presence in the gonad and adrenal gland invites the possibility of a wider role for PTHrP.

Calcium-dependent and calcium-independent exocytosis.

A large body of evidence supports the concept that calcium (Ca2+) plays a pivotal role in the control of exocytosis. However, recent experiments suggest that a rise in intracellular Ca2+ does not necessarily trigger secretion, and also that secretion can occur independently of cytosolic free calcium levels. This article briefly summarizes the early evidence that has formulated the role of Ca2+ in secretion, and then examines some of the recent evidence suggesting a Ca2+-independent mechanism of exocytosis.

A method for making solution changes in the sub-millisecond range at the tip of a patch pipette.

A method is described here for making multiple fast external solution changes at the tip of a patch pipette. The time for the change, 0.2 ms, has been established by measuring changes in liquid junction potential at the tip of an open patch pipette. This technique of producing an abrupt change in solution allows agonist/receptor reactions to be studied under non-equilibrium conditions. We have applied this technique to the nicotinic receptors in outside-out patches from skeletal muscle cell line C2 (Jaffe and Saxel 1979) and from bovine adrenal chromaffin cells. The application of step changes in acetylcholine concentration produces current traces with a characteristic shape, which may be compared with the predictions of established models for the activation and desensitisation of the nicotinic receptor. The results of making single steps and also short pulses in acetylcholine concentration are demonstrated. The direct comparison of two different cholinergic agonists is demonstrated.