ABSTRACT
Exocytosis of neurosecretory granules in the corpus cardiacum of the blowfly Calliphora erythrocephala was induced both by electrical stimulation and by depolarization with K+. This secretory activity was quantitatively determined by the frequency of omega figures and vesicle clusters, which are the ultrastructural indications of two distinct phases of the exocytotic process. Experimentally elicited exocytosis was reduced when Ca2+ were omitted from the bathing medium. This result supports the idea that the coupling between excitation (depolarization) and exocytosis involves Ca2+ influx through the axolemma. Barium effectively substitutes for calcium in the secretory process. High levels of magnesium in the bathing medium, however, decreased exocytosis, possibly by interfering with calcium influx.
INTRODUCTION
The corpus cardiacum neurosecretory cells (c.n.c.) of the blowfly Calliphora erythrocephala release the contents of their granules by exocytosis (Normann, 1965). The presence in thin sections of omega-like figures is the characteristic ultrastructural indication of this process. Omega figures are formed by fusion of granule membranes with the axolemma, the contents thus being voided and allowed to dissolve and escape by diffusion.
Clusters of small vesicles (30–40 nm) at the axolemma can also be observed in actively secreting axonal endings. These configurations were previously interpreted as indications of a different release mechanism, viz. intracellular fragmentation of granules. However, substantial evidence supports the idea that in neurosecretory cells ‘omega figures’ and ‘vesicle clusters’ represent consecutive phases of exocytosis viewed as a dynamic sequence of membrane phenomena (Normann, 1969). The microvesiculation provides for membrane retrieval and prevents addition of excess membrane area to the axolemma during secretion (Normann, 1970).
The same mechanism of neurosecretion operates in other animals (Bunt, 1969; U. Smith, 1970; Nagasawa, Douglas & Schulz, 1970; Santolaya, Bridges & Lederis, 1972; and others), and the view that exocytosis is the general release mechanism of neurohormones and perhaps all neurohumours is gaining acceptance (see A. D. Smith, 1971).
Calcium is known to be essential for stimulus-secretion coupling in neurosecretion and secretion by neurones (Douglas & Poisner, 1964; Douglas, 1966; Berlind & Cooke; 1968; reviews by Douglas, 1968; Simpson, 1968; A. D. Smith, 1971). However, the precise site of action of calcium remains to be identified (see Discussion).
The purpose of this study is to examine the role of calcium in experimentally elicited neurosecretion by exocytosis. The effects of increasing the magnesium concentration and of substituting barium for calcium in the medium have also been tested.
METHODS
Electrical stimulation
Corpora cardiaca of 4-day old blowflies were stimulated in situ as previously described (Normann, 1970). Prior to stimulation the organs were bathed for 3–4 min in one of the following fluids, which was repeatedly changed to wash away all haemolymph: Calliphora-Ringer (Normann, 1973) containing 5 mm calcium (‘R’), Ringer without added calcium (‘R–Ca’), or calcium-free Ringer containing 1 mm EGTA (βR–Ca EGTA’). EGTA (Ethyleneglycol-bis-(β-aminoethyl ether), N,N’-tetra-acetic acid) strongly binds calcium while negligibly affecting magnesium concentration.
The 3 groups (‘SR’) were subsequently stimulated for 1–2 min whilst being kept in the appropriate experimental fluid, which (after a total of 5 min) was replaced by fixative for electron microscopy. The controls (‘R’) were not stimulated, but were bathed in Ringer for 5 min.
Depolarization by high potassium
Three groups of corpora cardiaca were bathed for 3 min in Ringer containing calcium as indicated in the three pairs of histograms on the right of Fig. 1. This medium was subsequently replaced by a Ringer with similar concentration of calcium, but with potassium concentration increased from 5 to 100 mm. Glucose was omitted from the potassium-rich Ringer to avoid hypertonicity. After 1 min in the potassium-rich Ringer (‘R + K’), this was replaced by fixative. One group was treated similarly, except that the magnesium concentration had been increased from 2 to 20 mm (5 mm Ca2+). Another group was treated with a calcium-free Ringer containing 1 mm EGTA, to which 5 mm barium had been added.
Secretory activity was determined by electron microscopy as described in previous studies (Normann, 1969, 1970). The omega figures (Fig. 4, Plate 3; Fig. 5, Plate 4) and vesicle clusters (Fig. 6, Plate 4) were counted and their occurrence expressed as observed numbers per 100 axon profiles examined. In each corpus cardiacum at least 325 profiles of neurosecretory axon endings (400 on average) have been examined.
RESULTS
Some relevant morphological features of the corpus cardiacum are illustrated in Figs. 2 and 3 (Plates 1 and 2). (A more detailed structural analysis has been given previously (Normann, 1965)). Fig. 2, Plate 1, shows a lateral portion of the corpus cardiacum located between the aorta and the oesophagus. The c.n.c. bodies are situated at the ventral and lateral periphery of the gland, and axonal projections extend into the neuropile (Fig. 3, Plate 2).
Haemocoel indentations ramify deeply into the neuropile, thus facilitating exchange of substances between the latter and the blood. However, not all neurosecretory axon terminals face haemocoel spaces directly; in fact, exocytosis is most often observed at the narrow (10-20 nm) intercellular cleft between apposed neurosecretory axons or opposite a glial cell.
Since exocytosis, apparently, depends on the availability of extracellular Ca2+, these must be present in a more or less diffusible form throughout the intercellular spaces. Not knowing how readily this compartment exchanges Ca2+ with the incubation solution, a short experimental period was chosen to avoid adversely affecting cellular ultrastructure and yet ensure a partial washout of Ca2+.
The corpus cardiacum neuropile contains two main types of neurosecretory axon endings. The majority, the c.n.c. axons, contain granules most of which are oblong and range in size from 180 to 300 nm. According to Knowles (1967) this can be termed ‘A-type’ neurosecretion and is probably peptidergic. Using the traditional term ‘granule’ instead of ‘vesicle’ appears to be justified in view of the apparent rigidity of the contents, which consist in part of rod-like, tubular subunits in a paracrystalline array (Fig. 4, Plate 4). This substructure is masked in intact intra-axonal granules by a substance that stains heavily with lead, and disappears quickly at exocytosis.
Axons of ‘B-fibre’ type, possibly aminergic (Knowles, 1967), from the brain innervate the c.n.c. axons by synapses (Fig. 4, Plate 3). These extrinsic presynaptic axons contain small (80 – 120 nm) dense core vesicles and synaptic vesicles (Normann, 1970).
The present study deals with exocytosis from the intrinsic (c.n.c.) axon endings. The omega figures (Fig. 4, Plate 3; Fig. 5, Plate 4) and the vesicle clusters (Fig. 6, Plate 4) were counted and their frequency alculated as numbers per 100 profiles of axon endings.
The left part of the histogram (Fig. 1) shows the results of the quantitative electron microscopical analysis of the controls (‘R’) and of the three groups of electrically stimulated corpora cardiaca (‘SR’) exposed to different external calcium concentrations. Neurosecretion by exocytosis is obviously reduced at low calcium concentration. The visible cellular events following the establishment of connexion between granule membranes and plasma membranes are, however, unchanged at low calcium concentrations. Thus, calcium appears to be involved at the very beginning of exocytosis, either by taking part in establishing the physical connexion between membranes, or perhaps by liquefying the axoplasm and thus increasing the mobility of the granules.
A decreased external calcium concentration is known to reduce membrane stability and to increase spontaneous activity of excitable cells (review by Triggle, 1972). It may be mentioned that application of calcium-free Ringer, particularly when EGTA had also been added, led to vigorous trembling of the somatic musculature. Nevertheless, the inhibition of secretory activity could be due to a decreased excitability of neurosecretory neurones, rather than to a dependence on calcium entry of the coupling between excitation and exocytosis.
To test this possibility the influence of varying the calcium concentration on the induction of exocytosis by total depolarization with potassium has been examined (right part of Fig. 1).
The large number of omega figures produced by high potassium medium supports the idea that depolarization of the plasma membrane allows it to fuse with granule membranes, provided calcium ions are present. The different relation between omega figures and vesicle clusters observed in the depolarization experiments may be partly due to a more effective induction of exocytosis and to a longer persistence of the ‘omega phase’ relative to the duration of the microvesiculation phase (cf. Normann, 1970).
There is some indication, however, that calcium can interfere with microvesiculation. With high potassium and with calcium raised to 20 mm the occurrence of vesicle clusters is significantly lower than at an external calcium concentration of 5 mm. And in the ‘S’ experiments, in which at least some of the calcium must have been removed from the intercellular spaces by means of EGTA, the relation between vesicle clusters and omega figures appears slightly different from the other groups.
Since some vesicle clusters have been found in nearly all specimens, even after 5 min at low external calcium concentration, these vesicles may be the remnants of an exocytotic activity occurring prior to calcium deprivation.
Magnesium is known to antagonize calcium influx; raising the concentration of magnesium can interrupt synaptic transmission (Del Castillo & Engbaek, 1954), and neurohormone release can be antagonized by increased external magnesium concentration (Douglas & Poisner, 1964; Berlind & Cooke, 1971; and others). A group of corpora cardiaca was examined after incubation and subsequent potassium depolarization in Ringer with 5 mm calcium and with magnesium concentration raised from 2 to 20 mm. The average number of omega figures per 100 axon profiles recorded in 7 specimens was 1·6 ± 0·37 (s.E.) which is significantly (P < 0·001) lower than in group ‘R + K, 5 mm Ca’ (Fig. 1).
Certain divalent cations, including barium, can substitute for calcium in neurohormone release (Haller et al. 1965; Dicker, 1966; Berlind & Cooke, 1971). Five corpora cardiaca were treated with a calcium-free, EGTA-containing Ringer, to which 5 mm barium had been added. The average frequency of omega figures was found to be 8·1 ± 1·27 (s.E.) which is significantly higher (P < 0·001) than in group ‘R+K,-Ca, EGTA’ (Fig. 1).
DISCUSSION
The corpus cardiacum neurosecretory cells of the blowfly secrete a hyperglycaemic neurohormone (Normann & Duve, 1969; Vejbjerg & Normann, 1974) and its release, when induced by electrical stimulation, can be correlated with exocytotic activity (Normann, 1969, 1970). The c.n.c. are excitable (3-7 msec spikes) and they are innervated through synapses, the presynaptic axons coming from the brain (Normann, 1973).
Release of neurohormone from the c.n.c. occurs by exocytosis (Normann, 1965). In view of the neuronal characters of the neurosecretory cells and of the occurrence of Brownian movement of granules in the axon endings, it was suggested that exocytosis was regulated by nervous impulses, membrane fusion occurring only during depolarization. This scheme was further suggested to be the general mechanism of neurohormone secretion (Normann, 1965).
The products of protein-secreting cells are normally sequestered in membranelimited vesicles or granules until exocytosis. However, in the case of the vertebrate neurohypophysis the exocytosis theory was for long viewed with scepticism. This was partly because of the failure until more recently to obtain plain ultrastructural evidence such as omega figures in this tissue. But though substantial evidence now supports the extended exocytosis theory (see Introduction) (Nagasawa et al. 1970; Douglas, Nagasawa & Schulz, 1971; Santolaya et al. 1972; and others), reference should nevertheless be made to a variant of the still widely held theory of ‘molecular dispersion’ (for a survey, see Douglas et al. 1971). The ‘complex dissociation’ hypothesis (Thorn, 1970) is based on biochemical studies indicating that calcium ions may interfere competitively with the binding of neurohypophysial hormones to their carrier proteins, and it also assumes that vasopressin-neurophysin complexes can be located in two distinct compartments within axon endings.
According to the ‘complex dissociation’ hypothesis, the well-established significance of calcium for stimulus-secretion coupling lies in its ability to dissociate the carrier protein from the neurohormone that is present in an ‘easily releasable pool’ in the cytoplasmic matrix. The hormone would then have to travel through the axolemmal barrier, which would presumably have been rendered temporarily permeable. Stored neurosecretory material would subsequently have to be conveyed from the granules through their membranes to replenish the hypothetical pool in the cytoplasmic compartment.
From a cytological point of view this scheme is questionable. Sequestering the neuro-secretory substance within granule membranes, a function of the Golgi apparatus seems advantageous for the cells for the following reasons, (i) The substance is protected from destruction by cytoplasmic enzymes. (2) The cell maintains control over a substance which might interfere with normal cellular function, should it escape into the cytoplasm. (3) The substance can be conveniently transported in its native form through the axon and stored in its terminal, from which (4) it can be released in quantal packets as demanded.
Our results show that the initiation of exocytosis is calcium-dependent, i.e. the ion exerts its function in the coupling between depolarization and membrane attachment This is in line with the studies of Douglas and co-workers, of Berlind & Cooke (1968, 1971) and of Uttenthal, Livett & Hope (1971). The latter found that neurophysin and vasopressin were released in parallel by a calcium-dependent mechanism, that is compatible with exocytosis rather than with release by ‘complex dissociation’.
It may be significant that the conditions leading to release are the same in neurosecretory neurones and in, for example, cholinergic neurones, in which the following sequence has been suggested: depolarization → entry of calcium → acceleration of quantal release (Katz, 1971). Recent ultrastructural evidence supports the view that transmitter release occurs by exocytosis (Nickel & Potter, 1971; Heuser & Reese, 1973; Pysh & Wiley, 1974).
The actual site of calcium action remains to be identified; it could be located in or at the interior surface of the plasma membrane, on the granule membrane, or even in the axoplasm. Recent findings indicate that calcium having entered a depolarized axon terminal can liquefy the axoplasm and induce Brownian movement enabling the vesicles to reach the axolemma (Shaw & Newby, 1972; Piddington & Sattelle, 1974).
An earlier observation of frequent collisions between granules in vivid Brownian movement and the axolemma was made on corpora cardiaca which had been dissected out (Normann, 1965). The observed Brownian movement could therefore result from cellular damagef; nevertheless, the collisions with the axolemma did not lead to attachment of the granules to the plasma membrane. In this study the effect of potassium and calcium was not tested.
The electrophoretic properties of chromaffin granules have been studied by Matthews (1971), who calculated the net number of negative changes on the surface (one for every 11·4 square nm). Assuming a similar potential of the interior plasmalemmal surface, the granule membrane and the cell membrane would each have a boundary ionic layer about 0·75 nm thick. At a distance of 1·5 nm these boundary layers would set up a potential barrier which must be overcome for the granule to interact with the cell membrane. Calcium, however, is effective in decreasing the width of the boundary layer (as are other divalent cations). Further, reduction of the boundary layer of the granule membrane may not be necessary, because a transitory charge reversal from negative to positive of the inner surface of the cell membrane may occur at the same time as calcium influx and so remove the potential energy barrier to interaction (cf. Matthews, 1971).
Calcium ions alone cannot account for the membrane interactions leading to membrane fusion. Some sort of recognition process is required, perhaps existing in the form of specific proteins or ‘active sites’ on the interacting membranes. The existence of such sites was suggested by del Castillo & Katz (1957; see also Katz 1969). Electron microscopical evidence for the presence of specific calcium-binding sites on synaptic vesicles (one site per vesicle) has recently been obtained by Politoff et al. (1973).
The available evidence does not permit the identification of membrane specializations and/or site(s) of calcium action in the c.n.c. That an appreciable net influx of calcium occurs during secretion is suggested by the following unpublished observation: when c.n.c. of the blowfly and of the locust are forced to secrete continuously in vivo for several minutes, the mitochondria increase in size and number, and large matrix granules (up to 100 nm), probably indicative of calcium accumulation, develop. The mitochondria may have a buffering function, maintaining a low cytoplasmic calcium concentration (cf. Baker, 1972). Electron microscope microprobe analysis of calcium binding in mitochondria and other cell components is planned and may yield quantitative data required for further elucidation of the mechanism of secretion.
ACKNOWLEDGEMENTS
Part of this work was done at the Department of Zoology, University of Cambridge, during the tenure of a Wellcome-Carlsberg Travelling Research Fellowship.
REFERENCES
EXPLANATION OF PLATES
PLATE I
Fig. 2. Low magnification electron micrograph of a lateral portion of the corpus cardiacum, which is situated between the aorta and the oesophagus. The bodies of the corpus cardiacum neurosecretory cells, the c.n.c., are located at the ventral and lateral periphery of the ganglion-like gland. Most of the granule-containing axon profiles in the neuropile belong to the c.n.c. The axons that have few particles and are embedded in darker glioplasm belong to branches of the recurrent nerve. Note the deeply ramifying haemocoel indentations. AW, aorta wall; OES, oesophageal wall; CNC, c.n.c. bodies; HI, haemocoel indentations; NR, recurrent nerve, x 5850.
PLATE 2
Fig. 3. C.n.c. with axonal projection extending into the neuropile. N, nucleus of c.n.c.; A, axon, longitudinally sectioned; NP, neuropile; HI, haemocoel indentation. X7650.
PLATE 3
Fig. 4, Extrinsic microgranular axon of ‘B-type’ with synapse on a c.n.c. axon (‘A-type’). Otnega figure indicating exocytosis at arrow. In this specimen, Ba2+ had been substituted for Ca2+ prior to stimulation. x 60000.
PLATE 4
Fig. 5. Omega figures - exocytosis of neurosecretory granules from two adjacent axon terminals. Note the paracrystalline array of rod-like subunits, visible at the very beginning of exocytosis. This cannot be seen in intracellular (intact) granules (cf. text), x 98300.
Fig. 6. Vesicle clusters (VC), representing the process of membrane retrieval from the axolemma at release sites. Most of such microvesicles are 30-40 run (external diameter), x 154000.