F. C. G. van de Veerdonk

Morphological and physiological aspects of melanophores in primary culture from tadpoles of Xenopus laevis.

SummaryMelanophores from tadpoles of Xenopus laevis (Daudin) were isolated by digestion of tail fins with acetyltrypsin and collagenase and maintained in primary culture for 6 weeks up to 3 months. Within 36 to 72 h the melanophores develop one to eight dendritic processes per cell; secondary and tertiary branchings of the processes were frequently observed. The melanophores in primary culture disperse under the influence of α-MSH or cyclic AMP; upon rinsing out these substances the cells aggregate. In darkness, about 40 % of the cells disperse their pigment, whereas under illumination the pigment of the melanophores aggregates. To date, attempts to initiate cell division in melanophores have not been successful.

Role of calcium and prostaglandin (PGE1) in the MSH-induced activation of adenylate cyclase in Xenopus laevis

Abstract The effect of varying Ca 2+ concentrations on the action of a number of pigment dispersing substances has been studied. The absolute Ca 2+ requirement for MSH activity, contrary to the noradrenaline and cyclic AMP activities, suggests a role of Ca 2+ in the formation or the primary action of the MSH-receptor-complex. Prostaglandin (PGE 1 ) is also able to disperse the melanophores of Xenopus laevis. Its activity proves to be independent of the presence of Ca 2+ . A hypothesis has been put forward concerning the mechanism of MSH activity: formation of the MSH-receptor-complex may be responsible for prostaglandin synthesis in the membrane. This prostaglandin may produce displacement of Ca 2+ , followed by a Na + influx, which is responsible for the activation of the catalytic unit of membranous adenylate cyclase.

Demonstration of melatonin in amphibia

The presence of melatonin in the amphibian epiphysis has been ascertained earlier by several indirect methods, demonstrating the synthesizing enzyme or precursors of the compound. This communication describes the presence of melatonin in amphibian brain in a direct way, using dextran gel chromatography as a separation method.

Calcium sites in MSH stimulation of xenopus melanophores: Studies with photoreactive α-MSH

Abstract Photo-affinity labelling of MSH receptors on tail-fin melanophores of Xenopus tadpoles with p -azidophenylalanine 13 -α-MSH ([Pap 13 ]-α-MSH) or p -azidophenylacetyl-serine 1 -α-MSH ([Apac-Ser 1 ]-α-MSH) results in a long-lasting stimulation of the melanophores which cannot be reversed despite continuous washing. The generation of this irreversible response is inhibited when photo-affinity labelling is performed in a Ca 2+ -free medium or in the presence of Ca 2+ antagonists. The irreversible stimulation produced in normal medium is completely reversed upon removal of Ca 2+ , but is not affected by Ca 2+ antagonists or melatonin. Re-addition of Ca 2+ after temporary removal restores the irreversible stimulation, even in the presence of Ca 2+ antagonists or melatonin. This proves that covalent α-MSH-receptor complexes remain fully functional despite temporary deprivation of Ca 2+ . Racemized α-MSH, which binds ‘tightly’ to the receptor and produces a long-lasting effect, is removed from the receptor in Ca 2+ -free medium, but not by Ca 2+ antagonists or melatonin. These results confirm earlier results showing that at least 2 Ca 2+ sites are involved in α-MSH action, one associated with MSH-receptor binding and the other with the subsequent generation of the effect. The dual role of Ca 2+ is not the result of EGTA present; it is specific (Mg 2+ has no effect) and is identical for the two different photoreactive α-MSH derivatives.

Effect of injecting 5-methoxy indoles, pineal compounds, on testicular weight of white leghorn cockerels (Gallus domesticus L.)

White leghorn cockerels of the same age with combs of about equal size and raised under identical conditions were injected with melatonin, 5-methoxytryptophol, or with 5-methoxytryptophol in combination with melatonin. Melatonin, injected in animals with small comb sizes, has no effect on testicular growth, whereas an inhibiting effect is noticed in animals with larger comb sizes. The 5-methoxytryptophol treated animals demonstrated stimulated testicular growth, which can be interpreted as an acceleration of the rhythm of growth. The stimulatory effect on testicular growth is more distinct if 5-methoxytryptophol is injected in combination with melatonin.

Calcium requirement for α-MSH action on tail-fin melanophores of xenopus tadpoles

Abstract The role of Ca 2+ in α-MSH action on melanophores was studied, in vitro, with a bioassay on ventral tail-fin pieces from tadpoles of Xenopus laevis . Melanosome dispersion induced by α-MSH required 1–2 mM extracellular Ca 2+ . Gradual lowering of the extracellular Ca 2+ levels produced a concentration-dependent inhibition of the α-MSH response; complete inhibition was obtained in a Ca 2+ -free medium containing 10 −4 M EGTA. In Mg 2+ -free medium, normal dispersion was observed. The Ca 2+ antagonists verapamil (10 −4 M), methoxy-verapamil (10 −4 M) and La 3+ (10 −3 M) inhibited the dispersion induced by 3 × 10 −9 M α-MSH, whereas ruthenium red (10 −3 M) was without effect. The ionophore A23187 mimicked the effect of the hormone. Melanosome movement per se was evidently independent of Ca 2+ , because cAMP and dibutyryl-cAMP induced a full dispersion in the absence of Ca 2+ . These results show that extracellular Ca 2+ is specifically required for α-MSH action on tail-fin melanophores in vitro and suggests a Ca 2+ influx concomitant with the action of the hormone. Possible intra- and extra-cellular Ca 2+ sites are discussed.

A new in vitro melanophore bioassay for MSH using tail-fins of xenopus tadpoles

A new in vitro melanophore system is described, which employs pieces from the ventral tail-fin of Xenopus laevis tadpoles. Tail-fin melanophores in vitro retain the ability to disperse their pigment in darkness and to reaggregate it upon illumination. In the light, alpha-MSH, cAMP, dibutyryl-cAMP and theophylline induce a concentration-dependent pigment dispersion. The log dose-response curve obtained with alpha-MSH is sigmoidal with a linear portion between 0.5 and 2.0 ng alpha-MSH/ml. In this range, the log dose-response curve can be used as the standard curve in a bioassay for melanotropic activity, applying either the melanophore index (assay I) or a photometric transmittance measurement (assay II) for the quantification of the melanophore response. To prevent interference from the light/darkness response, light of 400-500 nm (to which the melanophores are most sensitive) was used during the assay. Both assays show high precision (lambda I = 0.13, lambda II = 0.11). Several peptides derived from alpha-MSH were tested for their melanotropic activity. The in vitro Xenopus melanophore system offers unique properties for the study of alpha-MSH action: (1) the melanophore system is uncontaminated with other chromatophores; (2) to date it is the only system suitable for photoaffinity labelling of alpha-MSH receptors; and (3) the melanophore receptor requirements differ from those of Rana.

A contribution to the analysis of the mechanism of MSH-induced dispersion in the melanophores of Xenopus laevis

The way in which MSH exerts its dispersing influence on the melanin granules in the melanocytes of amphibians has been the subject of many investigations. Different aspects of the dipersion process have been discussed by a number of investigators. The facts point to a rather complicated series of reactions, starting with the (hypothalamically regulated) secretion of MSH from the intermediate lobe of the pituitary gland into the blood stream, and resulting in the migration of the melanin granules from the central part into the processes of the melanocyte. The direct stimulus for the movement of the granules is probably related to a change in the electrolyte composition of the cytoplasm.

A fluorescent modification of the Sakaguchi reaction on arginine

SummaryThe reactionproduct of arginine with 2,4-dichloro-α-naphthol proved to be fluorescent. This fluorescence could be used for the histochemical localization of arginine. The modification described resulted in a long lasting yellow-orange fluorescence of various arginine-containing tissues.

Fluorescence of indole derivatives

Die Verwendung von 1N Natronlauge und Essigsäureanhydrid bei der Bestimmung von Indolderivaten mit Hilfe der Fluoreszenzmethode erlaubt eine gute Differenzierung zwischen 5-Methoxyindolderivaten (z.B. Melatonin) und anderen Indolen. Versuche zur Verwendung der neuen Methode in biologischem Material, z.B. Epiphysenschnitten, sind im Gange.