Niels A. Thorn

Rats with physically disconnected hypothalamo-pituitary tracts no longer contain vasopressin-oxytocin gene transcripts in the posterior pituitary lobe.

In rats, vasopressin‐ and oxytocin‐encoding mRNAs are present in the posterior but absent in the anterior lobe of the pituitary gland. RNase protection experiments indicate that in the posterior pituitary and hypothalamus identical transcriptional start points are used. Furthermore, the two transcripts from posterior pituitary and hypothalamus show identical nucleotide sequences. Animals operated by paired electrical lesions in such a way that connections between the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus and the posterior pituitary lobeare destroyed continue to express the vasopressin and oxytocin gene in the hypothalamus but not in the posterior pituitary. Operated animals subjected to chronic intermittent salt loading for 6 days similarly contain vasopressin and oxytocin encoding transcripts in the hypothalamus but not in the posterior pituitary.

Fusion of neurohypophyseal membranes in vitro.

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca2+ in micromolar concentrations. Mg2+ and Sr2+ were ineffective. Mg2+ inhibited Ca2+-induced fusion. In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion and fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca2+. Mg2+ and Sr2+ were ineffective in replacing Ca2+ as trigger for fusion or vasopressin release. Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosomes were exposed to cold.

Calcium and neurosecretion.

Neuroendocrine cells are excitable; i.e., a stimulation of them produces a plasma membrane polarization change of the type that is characteristic of nerve cells. However, they are very similar to pancreatic beta-cells in certain other aspects of their function since they release a polypeptide and since the release requires extracellular calcium. The studies by our group have centered on the neurohypophysis that releases vasopressin and oxytocin, two nonapeptide hormones. Dreifuss and his coworkers’ have also done a number of experiments on these nerve cells including studies of calcium movements over cell membranes. The hypothalamo-neurohypophyseal tract is a classical example of a neurosecretory system and most studies on the role of calcium in neurosecretion have been made on this system. The limited number of investigations reported from other neurosecretory systems seem to show that the phenomena that go on at hormone release from the neurohypophysis are typical for these systems in general. For example, it was demonstrated by Bennett & Edwardson’ that depolarizing concentrations of potassium release a corticotropin-releasing factor and a prolactin-release-inhibiting factor from isolated nerve endings (secretosomes) from rat hypothalamus in a calciumdependent manner. Warberg et al. showed that luteinizing-hormone-releasing hormone, thyrotropin-releasing-hormone, and a-MSH behave in a similar way. Neurosecretion has certain similarities to release of adrenalin and noradrenalin from the adrenal medulla. Poisner4 has given a review of the role of calcium mainly in that system, to which the reader is referred. A substantial amount of evidence supports the hypothesis that release of neurohypophyseal hormones occurs by e x o c y t ~ s i s . ~ ~ ~ Recently it has been demonstrated by freeze-cleaving electron microscopy that severely stimulated isolated neurohypophyses show exocytosis figures.’ This comprised fusion of secretory vesicles with the cell membrane. In addition, fusion of secretory vesicles with other vesicles undergoing exocytosis was observed (as well as fusion of secretory vesicles with each other) where no connection between vesicles and cell membrane was evident. In the experiment of Gratzl et al.’ it was demonstrated that strongly stimulated isolated neurohypophyseal nerve endings (secretosomes) show membrane alterations typical for exocytosis (“necks” and “holes”). It should, however, be mentioned that previously it has been stressed that certain findings could

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 microM free Ca2+ and a maximal uptake of 1.5 nmol X mg protein-1 X min-1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.

Parallel expression of synaptophysin and evoked neurotransmitter release during development of cultured neurons

Primary cultures of GABAergic cerebral cortex neurons and glutamatergic cerebellar granule cells were used to study the expression of synaptophysin, a synaptic vesicle marker protein, along with the ability of each cell type to release neurotransmitter upon stimulation. The synaptophysin expression and neurotransmitter release were measured in each of the culture types as a function of development for up to 8 days in vitro, using the same batch of cells for both sets of measurements to obtain optimal comparisons. The content and the distribution of synaptophysin in the developing cells were assessed by quantitative immunoblotting and light microscope immunocytochemistry, respectively. In both cell types, a close parallelism was found between the temporal pattern of development in synaptophysin expression and neurotransmitter release. This temporal pattern differed between the two types of neurons. The cerebral cortex neurons showed a biphasic time course of increase in synaptophysin content, paralleled by a biphasic pattern of development in their ability to release [3H]GABA in response to depolarization by glutamate or elevated K+ concentrations. In contrast, a monophasic, approximately linear increase in the synaptophysin content and stimulated [3H]D‐aspartate release was found in the cerebellar granule cells. These results, particularly regarding the GABAergic neurons, offer correlative evidence in support of the notion that a vesicular pool of these amino acid neurotransmitters may be intimately involved in their release, subsequent to depolarization stimuli.

HEXOSAMINE, CALCIUM, AND NEUROPHYSIN IN SECRETORY GRANULES AND THE ROLE OF CALCIUM IN HORMONE RELEASE

Vasopressin or oxytocin can be released from isolated halved neurohypophyses of rats or from slices of ox neurohypophyses by a 56 mM potassium medium or by electrical stimulation. Such experiments led to discussions about the existence of an “easily releasable pool” of hormone. The basis for this was that on prolonged stimulation only 5-10% of the vasopressin content of rat neurohypophyses could be released.’ Sachs and Haller2 found similar relations in in vivo and in vitro experiments on dogs in which vasopressin release was stimulated by hemorrhage. It is possible that, on stimulation, release preferentially takes place from a certain pool of hormone: but the results of recent experiments in our laboratory indicate that a substantial fraction of the vasopressin stored and even more so of oxytocin can be released when appropriate stimuli are applied. The results of some of these experiments are illustrated in FIGURE 1. I t can be seen that when sodium is omitted (replaced by sucrose) from the medium during incubation of groups of isolated halved rat neurohypophyses, a considerably larger release of vasopressin takes place than in a medium with a normal sodium concentration. This is also the case if sodium is removed late in a prolonged stimulation period, provided that Ca2+ is present. The total amount of vasopressin that could be released by such procedures was equivalent to some 20% of the total contents. Experiments on oxytocin release have given comparable results. In addition it has been demonstrated that treatment of isolated neural lobes with Nethylmaleimide (NEM) for 10 min (which seems to increase the intracellular concentration of free calcium): followed by a prolonged stimulation by 56 mM potassium, releases about 45% of the total tissue store of oxytocin (Dalby et al., in preparation). Further, at variance with the results of Sachs et al.; rat neurohypophyses stimulated repeatedly with a 56 mM K+ niedium with short intervals (of 12 min duration) of incubation in control (4.8 mM K+) medium, responded with at least a 5-fold increase in oxytocin release from baseline level even 210 min after the start of the stimulation. In a previous study6 it was found that addition of ATP plus an ATP regenerating system to preparations of isolated neurosecretory granules caused a release of vasopressin. These results have been extended (Russell and Thorn, in prepara-

Effect of hypoosmolality on the abundance, poly(A) tail length and axonal targeting of arginine vasopressin and oxytocin mRNAs in rat hypothalamic magnocellular neurons.

Arginine vasopressin (AVP) and oxytocin (OT) mRNAs are targeted to the axonal compartment of rat hypothalamic magnocellular neurons. Salt‐loading results in a considerable rise in hypothalamic and axonal AVP mRNA but only a moderate increase for axonal OT mRNA. Here we report that hypoosmolality gives rise to a rapid decrease of axonal AVP encoding transcripts to undetectable levels after 2 weeks. The levels of OT mRNA in the axonal compartment did not change significantly. In the hypothalamus the mRNA for AVP also decreased. The size of the poly(A) tract of AVP encoding transcripts appeared to be strictly correlated with plasma osmolality. In contrast, the amount and size of OT encoding mRNAs were only moderately or not influenced by hypoosmolar stimuli.

Ca2+ uptake to purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses were found to take up Ca2+ when incubated at 30 degrees C in media containing 10(-7) to 10(-4) M free Ca2+. At 10(-4) free Ca2+ 19 nmol/mg protein were taken up within 30 min. The initial uptake at this Ca2+ concentration was about 2 nmol/mg protein per min. The uptake of Ca2+ to secretory vesicles was not affected by ATP, oligomycin, ruthenium red, trifluoperazine, Mg2+ or K+, but was inhibited by Na+ and Sr2+. From these characteristics it can be concluded that the uptake system does not utilize directly ATP (as the Ca2+-ATPases known to be present in the cell membrane and the endoplasmic reticulum) and is different from the mitochondrial Ca2+ uptake system driven by respiration and/or ATP hydrolysis. However, Ca2+-Na+ exchange may well operate: In experiments using different concentrations of Na+ we found half-maximal inhibition of Ca2+ uptake with 33.3 mM Na+. An analysis of the data in a Hill plot indicated that at least 2 Na+ would be exchanged for 1 Ca2+. Also, it was found that Ca2+ previously taken up could be released again by external Na+ but not by K+.

Isolation and purification of calcium-binding proteins from bovine neurohypophyses

An acidic calcium-binding protein was isolated from the soluble fraction of the homogenate of ox neurohypophyses. The protein has a molecular weight of 35 000 and a subunit weight of 15 000. The purification procedure involved ammonium sulphate fractionation, DEAE-cellulose chromatography and gel filtration on Sephadex G-100 and Sephadex G-50. Conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated it to be a protein distinct from the S-100 protein and the soluble hormone-binding proteins (neurophysins) abundant in the neurohypophysis. This appears to be the only Ca2+-binding protein in the soluble part of the homogenate, with an apparent Kdiss for Ca2+ of 1.1 X 10(-5) M (at 22 degrees C) and a binding capacity of 2 mol of calcium per mol of protein. Two different Ca2+-binding proteins of molecular weights 16 500 and 68 000, respectively, were identified in the sodium-deoxycholate-soluble proteins from an ox neurohypophysial microsome fraction. One of them (the former) has been isolated in high purity by DEAE-cellulose chromatography and gel filtration on Sephadex G-200. This protein binds 4 mol of calcium per mol of protein with an apparent Kdiss of 1.0 X 10(-5) M (at 22 degrees C). The sodium-deoxycholate-insoluble proteins from the microsomal fraction also have Ca2+-binding components. The soluble Ca2+-binding protein has properties similar to and may be identical to Ca2+-binding proteins which have been isolated from bovine brain and have been demonstrated to be modulators of brain cyclic nucleotide phosphodiesterase and of actinomyosin ATPase. It also resembles Ca2+-binding proteins isolated from bovine adrenals and the electroplax from electrophorus electricus.

Stimulation by calmodulin of Ca2+ uptake and (Ca2+-Mg2+) ATPase activity in membrane fractions from ox neurohypophyses

Abstract Calmodulin stimulated 45 Ca 2+ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The 45 Ca 2+ uptake and the (Ca 2+ -Mg 2+ ) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca 2+ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca 2+ concentration in the neurohypophysis.