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 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.

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.

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.

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.

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.

Peter Baker was brought up in Lincoln where his father Frederick Thomas, always known as Tom, was curator of the Lincoln museum. Tom started work at the age of 16 and eventually became Director of the city museum, library and art gallery. His interests in local history and archaeology were recognized by election to the Fellowship of the Society of Antiquaries, by the award of an Honorary Degree at Hull University, and by the appointment as an Officer of the Civil Division of the Order of the British Empire. Tom married Doris Enid Skelton, a Lincolnshire girl, in 1937 and Peter, their only child, was born in 1939. Peter went to Lincoln School where he received a good all-round education, as well as a thorough grounding in science. In 1956 he won a major scholarship to Emmanuel College, Cambridge.

Comment Calcium and magnesium share a transport system in the gut, and both 1-a-hydroxycholecalciferol and 1,25 dihydroxycholecalciferol have been successfully used in the treatment of hypomagnesaemia associated with the short bowel syndrome.2 A recent report describes the use of l-a-cholecalciferol in a patient with the short bowel syndrome in whom, the authors claim, renal tubular absorption of magnesium was increased.3 In our case oral magnesium supplements in high doses were associated with diarrhoea, and I-a-hydroxycholecalciferol enhanced magnesium absorption and possibly reduced urinary losses. The figure shows that normal

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.

The role of sodium and calcium ions in the vasconstrictor response of isolated rat aorta and portal vein to synthetically prepared endothelin is investigated. Contractile responses to endothelin, unlike those induced by the sodium channel activator veratridine, are unaffected by tetrodotoxin or by the removal of sodium chloride from the solution bathing the tissue. The responses are the same whether sodium chloride is replaced iso-osmotically with either sucrose or potassium chloride. The endothelin responses in all media are entirely dependent on the presence of extracellular calcium, and can be blocked by 1 μM nitrendipine. These findings offer no support to the idea that voltage activated sodium channels are the primary site of action of endothelin as suggested by sequence homologies to scorpion α-toxins, but are entirely consistent with the possibility that the site of action of endothelin is closely coupled to the calcium channel (Yanagisawaet al, 1988).

Exocytosis, the process by which intracellular vesicles fuse with the inner surface of the plasma membrane, is thought to be the main mechanism underlying synaptic transmission at noradrenergic nerve terminals. A detailed study of the intracellular mechanisms involved has been hampered by the relative inaccesibility to the cytosol of markers that report intracellular chemical changes associated with secretion, or of solutes that clamp the chemical composition at the site of exocytosis. For this reason we have used not sympathetic nerve terminals as our experimental preparation, but instead the adrenal medullary (chromaffin) cell [1]. Although the medulla is part of the endocrine system, the cells are derived embryologically from neural crest tissue and retain the neuronal properties of excitable tissue, that is they exhibit sodium dependent action potentials and have voltage sensitive calcium channels [2]. One marked difference between an adrenal medullary cell and a sympathetic nerve terminal is the rate of secretion of transmitter. All the evidence suggests that noradrenaline is secreted form nerve terminals within milliseconds of the arrival of an action potential, whereas secretion from the adrena medullary cell proceeds at a much slower rate [3]. Apart from this difference the adrenal medullary cell serves as an excellent model of a sympathetic neurone in which to study the mechanism of catecholamine secretion, and this short article reviews the experimental approaches we have used here at King’s College, the results and conclusions reached, and the direction we will be taking in the future.

The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca2+ levels much in excess of 50 μm. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca2+ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.

Measurements of intracellular Ca2+ in adrenal medullary cells suggest that a transient rise in Ca2+ leads to a transient secretory response, the rise in Ca2+ being brought about by an influx through voltage-sensitive Ca channels which subsequently inactivate. The level of Ca2+ observed is much smaller than the Ca2+ needed to trigger secretion when introduced directly into the cell. The discrepancy is removed by the presence of diacylglycerot, which increases the sensitivity of the secretory process to Ca2+. The site of action of Ca2+ and diacylglycerol is probably protein kinase C, and tile different secretory responses to increases of Ca2+ and diacylglycerol can be modelled in terms of a preferential order of binding of these two substrates to the enzyme. ATP is needed for secretion: one role is possibly to confer stability to the secretory apparatus; another may involve phosphorylation of some key protein. The kinetics of secretion suggest that if Ca2+ regulates phosphorylation or dephosphorylation, then it is therate of change of phosphorylation that controls secretion rather than theextent of phosphorylation or dephosphorylation. Guanine nucleotide-binding proteins may play a role not only at the level of signal transduction coupling, but also at or near the site of exocytosis, and the mechanism by which some Botulinum toxins inhibit secretion may be associated with these proteins.

Annals of the New York Academy of SciencesVolume 493, Issue 1 p. 504-523 Exocytosis from the Vesicle Viewpoint: An Overview D. E. KNIGHT, D. E. KNIGHT Medical Research Council Secretory Mechanisms Group, Department of Physiology Kings College Strand, London WC2 R2LS, United KingdomSearch for more papers by this authorP. F. BAKER, P. F. BAKER Medical Research Council Secretory Mechanisms Group, Department of Physiology Kings College Strand, London WC2 R2LS, United KingdomSearch for more papers by this author D. E. KNIGHT, D. E. KNIGHT Medical Research Council Secretory Mechanisms Group, Department of Physiology Kings College Strand, London WC2 R2LS, United KingdomSearch for more papers by this authorP. F. BAKER, P. F. BAKER Medical Research Council Secretory Mechanisms Group, Department of Physiology Kings College Strand, London WC2 R2LS, United KingdomSearch for more papers by this author First published: April 1987 https://doi.org/10.1111/j.1749-6632.1987.tb27237.xCitations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume493, Issue1Cellular and Molecular Biology of Hormone- and Neurotransmitter-Containing Secretory VesiclesApril 1987Pages 504-523 RelatedInformation

Calcium-dependent exocytosis in 'leaky' bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in 'leaky' cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measured in situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.