Correlation of the number of pineal “synaptic” ribbons and spherules with the level of serum melatonin over a 24-hour period in male rabbits

Summary Previous studies have shown that pineal synaptic ribbons and spherules may respond differently under normal and experimental conditions. It has been suggested that the increase in the number of ribbons may be a prerequisite for enhanced melatonin formation. In the present study, the number of ribbons and spherules as well as the level of serum melatonin were monitored over a 24-h period in the male rabbit, the pineal gland of which is known to contain many spherules. It was found that both the number of ribbons and the levels of serum melatonin show the typical nocturnal increase, exhibiting peaks at 02:00 and 06:00 h, respectively. There is a good correlation (R = 0.8) of the two parameters. The spherules, in contrast, show no statistically significant circadian changes in number and cannot be correlated with the levels of serum melatonin. It is concluded that ribbons and spherules may differ in function and that the ribbons may be somehow involved in the regulation of melatonin formation.Supported by the Deutsche Forschungsgemeinschaft (Grant Vo 135/7) within the project SPP Neuroendokrinologie     

Depressive effect of LHRH on the numbers of “synaptic” ribbons and spherules in the pineal gland of diestrous rats

Summary Previous studies have shown that LHRH or LHRH-like substances are present in the pineal gland. In order to investigate whether exogenous LHRH may affect the pineal gland, in the present study the effects of a single dose of LHRH (1 g, i.p.) on pineal synaptic ribbons and spherules as well as serum melatonin levels were examined in diestrous Wistar rats. One hour after the injection both ribbons and spherules exhibited a statistically significant decrease in number. Serum melatonin levels were not affected. It is concluded that humoral feedback mechanisms may exist between the hypothalamus and the pineal gland.Supported by grant Vo 135/7 within the Schwerpunktprogramm Neuroendokrinolgoie of the Deutsche Forschungsgemeinschaft     

The effects of short pulses of light at night on numbers of pineal “synaptic” ribbons and serotonin N-acetyltransferase activity in male Sprague-Dawley rats

Summary To characterize further the functionally enigmatic synaptic ribbons (SR) of the mammalian pineal gland and to study possible relationships to melatonin synthesis, in the present investigation rats were exposed to short pulses of light at night when both SR numbers and serotonin N-acetyltransferase (NAT) activity are high in comparison to day-time values. Male Sprague-Dawley rats were killed at 13:00 and 01:00 h, respectively, and at 01:10 and 02:00 h after exposure to light for 10 and 60 min, respectively. The pineals were rapidly taken out and cut sagittally in half. One half was processed for electron-microscopic quantitation of SR numbers and the other half for NAT determinations. It was found that both SR numbers and NAT activity decreased significantly when the animals were exposed to light at night. Although both parameters showed corresponding changes, there was no clear-cut correlation between SR numbers and NAT activity in individual animals within a group, except after exposure to light for 60 min when a positive correlation (R = 0.939; p < 0.05) existed. After exposure to light the electron-lucent vesicles of the SR decreased in number, but the length of the SR was unchanged. These results show that numbers of pineal SR can be easily and quickly manipulated and that the presently used model may be ideal in studying the poorly understood mode in which degradation of SR occurs.Recipient of a DAAD stipend, on leave from Department of Zoology, University of Burdwan, Burdwan, India     

Release and effect ofγ-Aminobutyric acid (GABA) on rat pineal melatonin productionin vitro

1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures. 8. GABA at concentrations of 10(-5) M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants. 9. The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release. 10. The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels. 11. These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction with alpha 1-adrenoceptors; (c) GABA inhibits melatonin production in vitro via interaction with GABA type A receptor sites; and (d) GABA's effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.     

Day-night differences in the number of pineal “synaptic” ribbons in two diurnal rodents,the chipmunk (Tamias striatus) and the ground squirrel (Spermophilus richardsonii)

Summary Daytime numbers of pineal synaptic ribbons higher than reported in the pineal gland of any other mammalian species were observed in two diurnal rodents, the eastern chipmunk and Richardson's ground squirrel. The number of synaptic ribbons was lower during the daytime and higher at night in both of these species.     

Lack of effect of oxytocin on the numbers of “synaptic” ribbons,cyclic guanosine monophosphate and serotonin N-acetyltransferase activity in organ-cultured pineals of three strains of rats

In addition to the stimulating influence of the sympathetic system on the function of the mammalian pineal gland, neuropeptides such as neuropeptide Y, vasoactive intestinal polypeptide and arginine-vasopressin (AVP) are thought to function as modulators. Since AVP has been shown to influence pineal melatonin synthesis, the aim of the present study was to investigate the possible effects of the second hypothalamic nonapeptide oxytocin (OT), which likewise has been detected in the pineal gland. We therefore studied synaptic ribbon (SR) numbers, N-acetyltransferase (NAT) activity and the intracellular concentration of cyclic guanosine monophosphate (cGMP) following in vitro incubation of rat pineals in media containing OT (10^-5 M), noradrenaline (NA, 10^-5 M) or both NA and OT. Pineal glands were derived from rats of three different strains (Sprague-Dawley, Long-Evans and the AVP-deficient strain Brattleboro). Neither morphological nor biochemical analyses showed a difference between control and OT-incubated organs in any of the strains tested. In Brattleboro rats, but not in the other strains, noradrenaline slightly increased the number of SR which was not observed when NA and OT were combined. The addition of NA resulted in distinct augmentation of NAT activity and cGMP content, which were not affected by additional OT application. These results suggest that oxytocin is not crucially involved in the regulation of pineal gland function.     

Circadian variations of “synaptic” bodies in the pineal glands of Brattleboro rats

Summary The function of the mammalian pincal gland is regulated primarily by the sympathetic system. Arginine-vasopressin (AVP) may also be involved in the regulation of pineal melatonin synthesis under experimental conditions. The present study was conducted in the AVP-deficient rat strain, the Brattleboro rat, to investigate whether the numbers and rhythms of pineal synaptic bodies in this strain are different from those found in intact rats. AVP or its non-vasoconstrictive analog, deamino-D-AVP, was also injected intra-arterially in Brattleboro or Sprague-Dawley rats to test whether this procedure influences synaptic body numbers. Brattleboro rats were killed at different timepoints throughout the 24 h-cycle in March, June and September. Synaptic ribbons, spherules and intermediate structures were quantified and examined with regard to their intracellular location, with or without nocturnal AVP or D-AVP treatment. Numbers of ribbons were low during the day and high during the night (as in genetically intact rats), whereas spherules and intermediate structures numbers exhibited inconstant daily patterns. Night levels of synaptic ribbons were highest in June, lowest in March, whereas day levels did not differ significantly. No significant alterations in pineal synaptic body numbers were found following administration of AVP or D-AVP. Our results therefore indicate that AVP does not play a crucial role in the regulation of pineal synaptic body numbers in rats.     

Do thyroid and testis modulate the effects of pineal and melatonin on haemopoietic variables inClarias batrachus?

Involvement of pineal and its major hormone, melatonin, in the process of erythropoiesis in a freshwater catfish,Clarias batrachus has been investigated. The study was conducted during four phases, namely preparatory phase, spawning phase, postspawning phase and late postspawning phase of its annual reproductive cycle. During each phase a fish received either melatonin injections or subjected to pinealectomy. In addition, each fish in all the groups, received either iopanoic acid or cyproterone acetate or vehicle in the morning or late afternoon. Results clearly indicate that melatonin stimulates the rate of erythropoiesis inClarias batrachus. It appears that the extent of stimulation depends upon the phase of the annual reproductive cycle. However, in general, the pineal-or melatonin-induced modulation of blood variables is gonad dependent and thyroid seems to play a time of the day dependent subtle role     

Sitagliptin therapy enhances the number of circulating angiogenic cells and angiogenesis—evaluations in vitro and in the rat critical limb ischemia model

Background aimsWe tested the hypothesis that sitagliptin is capable of increasing blood flow in the rat critical limb ischemia (CLI) model by enhancement of angiogenesis.MethodsAdipose tissue from adult-male Fischer 344 rats (n = 6) were cultured in endothelial progenitor cell culture medium for 14 d with (25 μmol/L) or without sitagliptin. CLI was induced by ligation of the left femoral artery. Rats (n = 32) were equally separated into four groups: untreated controls (group 1), sitagliptin (4 mg/kg per day; group 2), CLI (group 3) and CLI with sitagliptin (group 4).ResultsIn vitro, 7 and 14 d after cell culture, endothelial progenitor cell biomarkers assessed by flow cytometry (Sca-1/CD31+, CXCR4+, c-kit+ and CD34+ cells) and Western blot (vascular endothelial growth factor, CXCR4 and stromal-derived factor [SDF]-1α) were remarkably higher in group 4 than in the other groups (all P < 0.01). In vivo, 2 and 14 d after the CLI procedure, circulating angiogenic cell (Sca-1/CD31+, Sca-1+ and CD31+) numbers were significantly higher in group 4 than in the other groups (all P < 0.001). Additionally, the messenger RNA and protein expression of angiogenic biomarkers (CXCR4, SDF-1α and vascular endothelial growth factor), immunofluorescent staining of angiogenic cells (CXCR4+, SDF-1α+, CD31+, von Willebrand factor + cells) and immunohistochemical staining of small vessel numbers in the ischemic area were significantly higher in group 4 than in the other groups (all P < 0.01). Furthermore, laser Doppler showed that the ratio of ischemic/normal blood flow was remarkably higher group 4 than in group 3 by days 14 and 28 after the CLI procedure (all P < 0.01).ConclusionsSitagliptin therapy enhances circulating angiogenic cell numbers, angiogenesis and blood flow in the CLI area.     

The structural and functional consequences of melatonin and serotonin on human αB-crystallin and their dual role in the eye lens transparency

Crystallins are the major soluble lens proteins, and α-crystallin, the most important protective protein of the eye lens, has two subunits (αA and αB) with chaperone activity. αB-crystallin (αB-Cry) with a relatively wide tissue distribution has an innate ability to interact effectively with the misfolded proteins, preventing their aggregation. Melatonin and serotonin have also been identified in relatively high concentrations in the lenticular tissues. This study investigated the effect of these naturally occurring compounds and medications on the structure, oligomerization, aggregation, and chaperone-like activity of human αB-Cry. Various spectroscopic methods, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and molecular docking have been used for this purpose. Based on our results, melatonin indicates an inhibitory effect on the aggregation of human αB-Cry without altering its chaperone-like activity. However, serotonin decreases αB-Cry oligomeric size distribution by creating hydrogen bonds, decreases its chaperone-like activity, and at high concentrations increases protein aggregation.     

Circadian-related heteromerization of adrenergic and dopamine D₄ receptors modulates melatonin synthesis and release in the pineal gland

The role of the pineal gland is to translate the rhythmic cycles of night and day encoded by the retina into hormonal signals that are transmitted to the rest of the neuronal system in the form of serotonin and melatonin synthesis and release. Here we describe that the production of both melatonin and serotonin by the pineal gland is regulated by a circadian-related heteromerization of adrenergic and dopamine D₄ receptors. Through α_{1 B}-D₄ and β₁-D₄ receptor heteromers dopamine inhibits adrenergic receptor signaling and blocks the synthesis of melatonin induced by adrenergic receptor ligands. This inhibition was not observed at hours of the day when D₄ was not expressed. These data provide a new perspective on dopamine function and constitute the first example of a circadian-controlled receptor heteromer. The unanticipated heteromerization between adrenergic and dopamine D₄ receptors provides a feedback mechanism for the neuronal hormone system in the form of dopamine to control circadian inputs.     

Neuroprotective effect of the hexapeptide HLDF-6 on rat hippocampal neurons on the in vivo and in vitro models of alzheimer’s disease

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer’s disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by injection of beta-amyloid peptide (25–35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the exploratory behavior of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of the rat hippocampal and cerebellar neurons under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer’s disease and in the medium of the culture of hippocampal neurons in the presence of the Aβ(25–35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

Hidden prenatal malnutrition in the rat: role of β₁-adrenoceptors on synaptic plasticity in the frontal cortex

Moderate reduction in the protein content of the mother's diet (hidden malnutrition) does not alter body and brain weights of rat pups at birth, but leads to dysfunction of neocortical noradrenaline systems together with impaired long-term potentiation and visuo-spatial memory performance. As β?-adrenoceptors and downstream protein kinase signaling are critically involved in synaptic long-term potentiation and memory formation, we evaluated the β?-adrenoceptor density and the expression of cyclic-AMP dependent protein kinase, calcium/calmodulin-dependent protein kinase and protein kinase Fyn, in the frontal cortex of prenatally malnourished adult rats. In addition, we also studied if β?-adrenoceptor activation with the selective β? agonist dobutamine could improve deficits of prefrontal cortex long-term potentiation presenting these animals. Prenatally malnourished rats exhibited half of β?-adrenoceptor binding, together with a 51% and 65% reduction of cyclic AMP-dependent protein kinase α and calcium/calmodulin-dependent protein kinase α expression, respectively, as compared with eutrophic animals. Administration of the selective β? agonist dobutamine prior to tetanization completely rescued the ability of the prefrontal cortex to develop and maintain long-term potentiation in the malnourished rats. Results suggest that under-expression of neocortical β?-adrenoceptors and protein kinase signaling in hidden malnourished rats functionally affects the synaptic networks subserving prefrontal cortex long-term potentiation. β?-adrenoceptor activation was sufficient to fully recover neocortical plasticity in the PKA- and calcium/calmodulin-dependent protein kinase II-deficient undernourished rats, possibly by producing extra amounts of cAMP and/or by recruiting alternative signaling cascades.     

Presence of AVT-, α-MSH-, LHRH- and somatostatin-like compounds in the rat pineal gland and their relationship with the UMO5R pineal fraction

Using antibodies against AVT, alpha-MSH, LHRH and somatostatin, immunoreactive cells were detected in the rat pineal gland. All of these antibodies stain the same cells, which also react immunocytochemically when an antibody against the UMO5R sheep pineal fraction, a fraction that presents antigonadotropic properties in vivo, is used. Relatively more immunoreactive cells are present in the pineals of young rats than in the pineals of adult animals. Comparison of the results obtained with different potent antibodies against each of the peptides, and a study of the staining properties of the antibodies in the pineal after solid phase absorption to different peptides or to different sheep pineal fractions, led to the proposal that the immunoreactivity found in the rat pineal is not due to the presence of AVT, alpha-MSH, LHRH or somatostatin, but to a cross-reaction of each of these antibodies with (an) unidentified compound(s). This compound is synthetized in the pineal gland, as was demonstrated using cultured pineals. The UMO5R and the Prot. 4 fractions of the sheep pineal seem to be chemically related to this unknown compound, the possible endocrine nature of which is discussed.     

Attenuation of morphine analgesia by α-MSH,MIF-I,melatonin and naloxone in the rat

In two experiments the effects of the pituitary peptide α-MSH, the hypothalamic tripeptide MIF-I (P-L-G-NH₂) and the pineal hormone melatonin were investigated on the attenuation of morphine analgesia measured by a tail flick test. In Experiment 1, α-MSH had minimal effect on morphine analgesia, whereas, MIF-I and melatonin clearly delayed the onset of morphine analgesia, and melatonin also shortened the duration of analgesia. Experiment 2 was designed to investigate the possible synergistic effect of MIF-I and melatonin. The combined treatment of MIF-I and melatonin significantly delayed the onset of morphine analgesia, and melatonin alone shortened the duration of analgesia. The relationshps among the pituitary, hypothalamus and the pineal for the modulation of pain and response to morphine were discussed.