Sol Oaknin

Age-related changes in 2-(125I)-Iodomelatonin binding sites in the brain of sea breams (Sparus aurata, L.)

The pineal organ of fish, through its 24h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g., reproduction, growth). The target sites for melatonin are poorly known in fish, especially marine species. A radioligand study was undertaken using the gilthead sea bream (Sparus aurata) maintained under natural temperature and photoperiod (at 28°N latitude). This species exhibits the property of changing sex during growth. Brains of one year-old males were collected at 16:00h and brains of three year-old females at 03:00, 10:00, 16:00 and 23:00h. Membrane homogenate receptor assays were run using 2-[125I]iodomelatonin as a ligand. Binding sites were detected in brains of young and old fish. In the younger, the exhibited a Bmax between 3.52 and 4.29 fmol mg protein−1 and a KD between 358–380 pmol l−1. In the older fish, the KD varied according to a daily pattern: values were three times higher at 03:00 and 10:00h (500–600 pmol l−1) than at 16:00 and 23:00h (150–300 pmol l−1). The number of sites also were higher at 03:00 and 10:00h (180–200 fmol mg protein−1) than at 16:00 and 23:00h (95–110 fmol mg protein−1). Melatonin and iodomelatonin displaced 2-[125I]iodomelatonin binding in a dose dependent manner, the second being more potent than the first. Binding was also inhibited by GTP. The results provide the first evidence for the presence of membrane melatonin binding sites in the brain of an exclusively marine fish. They suggest that their number and affinity varies during growth and throughout a light/dark cycle. Future experiments will aim to precise the anatomical location and role of these binding sites.

Receptor binding properties of di (1,N6-ethenoadenosine) 5′, 5′′′-P1, P4-tetraphosphate and its modulatory effect on extracellular glutamate levels in rat striatum

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.

Potent inhibition of specific diadenosine polyphosphate hydrolases by suramin

The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap4Aase) and diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap3Aase) are inhibited competitively by suramin. Ap4Aase and Ap3Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). K i values for suramin as inhibitor of Ap4Aase and Ap3Aase were 5×10−6 M and 3×10−7 M, respectively. Results indicate that suramin or suramin‐like derivatives may be useful tools to investigate diadenosine polyphosphate cleaving enzymes and that the intracellular diadenosine polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.

Changes in forebrain Na,K-ATPase activity and serum hormone levels during sexual behavior in male rats

We have previously shown that Na,K-ATPase activity (the enzymatic machinery for the sodium pump) in brain areas such as the medial basal hypothalamus (MBH) and the preoptic-suprachiasmatic region (POSC) can be changed by experimental manipulations of gonadal function. We now report enzyme levels in brain regions as related to hormonal changes occurring during sexual behavior. Male rats were exposed to receptive females and decapitated immediately after displaying one of the following behavioral events: the start of copulatory activity, first ejaculation, and the beginning of a second copulatory series. A group of noncopulating animals were used as control. The variables measured included serum levels of LH, PRL and testosterone and Na,K-ATPase activity in MBH, POSC and parietal cerebral cortex (CC). A steady increase in enzyme activity in the POSC, but not the MBH or CC, was found in copulating animals. Serum LH levels changed in a similar fashion. A sharp increase in serum PRL levels, seemingly related to ejaculation, was also observed. These data are consistent with our previous findings on monoaminergic neurotransmission in brain regions related to male sexual behavior.

Fluorimetric detection of enzymatic activity associated with the human tumor suppressor Fhit protein.

The human tumor suppressor Fhit protein exhibits diadenosine triphosphatase activity, hydrolyzing Ap(3)A to AMP and ADP. We report that Fhit protein efficiently cleaves the fluorogenic Ap(3)A analog diethenoadenosine triphosphate giving support to establish a simple fluorimetric assay for quantification of Fhit enzyme. Fluorimetric assays were initially tested to demonstrate that diethyl pyrocarbonate and suramin inhibit Fhit enzyme.

Injections of α-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.