Biomedical subjects

R Spessert

Publications and source records attributed to R Spessert.

28 records · Page 2Linked to original sources

PubMed1994

NADPH-diaphorase activity of nitric oxide synthase in the olfactory bulb: co-factor specificity and characterization regarding the interrelation to NO formation.

The neuronal form of the enzyme nitric oxide synthase (nNOS) synthesizes the messenger molecule nitric oxide (NO). In addition to NO formation, nNOS exhibits a so-called NADPH-diaphorase (NADPH-d) activity. This study focused on the characterization of NADPH-d activity with regard to NO formation in the rat olfactory bulb. In this area of the brain pronounced staining is localized in discrete populations of neuronal somata and in olfactory glomeruli. Diaphorase staining combined with demonstration of nNOS by polyclonal antibodies revealed that NADPH-d activity of neuron somata is associated with nNOS immunoreactivity. It is concluded that neuron somata exhibit NADPH-d activity of nNOS. NADPH-d activity of nNOS did not utilize beta-NADH or alpha-NADPH. Moreover, NADPH-d activity was inhibited in the presence of alpha-NADPH. Dichlorophenolindophenol (DPIP), an artificial electron acceptor and an inhibitor of NO formation, totally suppressed NADPH-d staining of neurons, supporting the concept that the NADPH-d of neuron somata is due to nNOS. Cytochrome C, miconazole, EGTA, and trifluoperazine, which have been reported to inhibit cytochrome P450 reductase activity of NOS, did not affect NADPH-d staining. Hence, NADPH-d activity of NOS does not involve cytochrome P450 reductase activity as required for NO formation. Contrary to NADPH-d activity of neuron somata, staining of olfactory glomeruli was not co-localized with nNOS immunoreactivity. Glomerular staining was also observed in the presence of beta-NADH and alpha-NADPH. Further, it was unchanged in the presence of the NO formation inhibitor DPIP. Hence, the glomerular staining in the presence of NADPH is not due to the NADPH-d activity of NOS. We conclude that staining of neuronal structures in the presence of NADPH does not necessarily represent NADPH-d activity of NOS.

2,6-Dichloroindophenol↗

PubMed1993

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.

Animals↗

PubMed1993

Muscarinic agonists have no measurable effect on cGMP formation of rat pinealocytes.

Previous studies have shown that muscarinic agonists stimulate cGMP formation in various tissues including rat brain. As in the pineal gland cGMP formation varies considerably under various experimental conditions, in the present investigation the effects of muscarinic agonists were tested. Muscarinic agonists neither stimulated pineal cGMP formation nor affected cGMP accumulation, resulting from administration of phosphodiesterase (PDE) inhibitors, norepinephrine (NE), or sodium nitroprusside (SNP). Because muscarinic agonists are known to stimulate pineal inositol phosphate (Ip) formation we suspect that muscarine-related Ip formation does not affect cGMP formation in rat pineal gland.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Adrenergic stimulation of cyclic GMP formation requires NO-dependent activation of cytosolic guanylate cyclase in rat pinealocytes.

Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving beta- and alpha 1-adrenergic receptors. The effects of NG-monomethyl-L-arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited beta-adrenergic stimulation of cGMP formation as well as alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of L-arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.

Animals↗

PubMed1993

Vasoactive intestinal peptide stimulation of cyclic guanosine monophosphate formation: further evidence for a role of nitric oxide synthase and cytosolic guanylate cyclase in rat pinealocytes.

In the rat pineal gland vasoactive intestinal peptide (VIP) and beta-adrenergic agonists stimulate cyclic guanosine monophosphate (cGMP) formation and their action is amplified by alpha 1-adrenergic agonists. Since beta-adrenergic stimulation of cGMP is suggested to involve activation of nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase (GC), we investigated the effects of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA) and of the cytosolic GC inhibitor methylene blue (MB) on VIP receptor-stimulated cGMP formation. Both L-NMMA and MB depressed VIP-induced cGMP formation as well as alpha 1-adrenergic potentiation of VIP-stimulated cGMP formation to the level of unstimulated pinealocytes. Further, L-arginine (L-arg) antagonized the effect of L-NMMA. However, L-arg did not antagonize the effect of MB, indicating that activation of NO synthase does not appear to compensate inhibition of cytosolic GC. On the basis of these findings it is concluded that VIP-stimulated cGMP response requires NO synthesis followed by activation of cytosolic GC. Major similarities between the regulation of VIP- and beta-adrenergic-induced cGMP formation suggest a similar/common intracellular pathway which can be modulated by alpha 1-adrenergic stimulation.

Amino Acid Oxidoreductases↗

PubMed1992

Involvement of cyclic guanosine monophosphate (cGMP) and cytosolic guanylate cyclase in the regulation of synaptic ribbon numbers in rat pineal gland.

In the rat pineal gland N-acetyltransferase (NAT) activity and synaptic ribbon (SR) numbers display a circadian rhythm. It is well-known that NAT activity is regulated by adrenergic mechanisms involving cyclic adenosine monophosphate (cAMP) as a second messenger. However, the mechanism involved in the regulation of SR numbers has not been established so far. In the present in vitro study, we have investigated the effects of 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), a cyclic guanosine monophosphate (cGMP) analog, and stimulation of guanylate cyclase on SR numbers. Incubation with 8-bromo-cGMP increased SR numbers in a dose- and time-dependent manner. Further, stimulation of the cytosolic guanylate cyclase also resulted in increased SR numbers. Adrenergic agonists stimulated cGMP but did not alter SR numbers. These findings suggest that cGMP is involved as a second messenger in the regulation of SR numbers. Since the adrenergically stimulated increase in cGMP did not influence SR numbers, a non-adrenergic cGMP metabolic pathway seems to be involved in the regulation of SR numbers in the rat pineal gland.

Animals↗

PubMed1992

In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland.

The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide (DSIP), vasoactive intestinal polypeptide (VIP), adenosine and N-acetyl-asp-glu (NAAG) significantly increased NAT activity in rat pineal. DSIP and VIP also increase the stimulatory effect of NE on NAT activity. These neurochemicals had no effect on SR numbers. Gamma aminobutyric acid (GABA), serotonin and taurine affected neither NAT activity nor SR. Somatostatin increased SR numbers significantly, without having any effect on NAT activity. The effect of somatostatin is regarded to be pharmacologic, since rather high dosages (10(-4) M) were required to obtain a significant effect. Although somatostatin is present in the pineal and may change rhythmically, the inconsistency of the day/night rhythmicity and the lack of such a rhythm in female rats and male gerbils speaks against an important physiological role of somatostatin in regulating SR numbers.

Animals↗

PubMed1992

Biphasic 24-hour variations in cyclic GMP accumulation in the rat pineal gland are due to corresponding changes in the activity of cytosolic and particulate guanylate cyclase.

Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lights "on" and the other approximately 7 h after lights "off." The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine, indicating that 24-h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.

Animals↗

PubMed1991

Acetylcholine and muscarinic agonists increase synaptic ribbon numbers in the rat pineal.

Mammalian pinealocytes possess synaptic ribbons (SR) which are commonly present in photoreceptor cells at synaptic junctions. Pineal SR numbers undergo a diurnal rhythm parallel to that of pineal N-acetyltransferase (NAT) activity and melatonin levels. Recent findings suggest that SR numbers, unlike NAT activity and melatonin synthesis and release, do not seem to be regulated by adrenergic mechanisms or neuropeptides in adult rats. Since the pineal gland also receives cholinergic nerve fibres, we have investigated in vitro effects of acetylcholine (ACh) and carbamyl-beta-methylcholine (CBMC; a specific muscarinic agonist) in the presence and absence of pirenzipine (a specific inhibitor of muscarinic M1 receptors). ACh and CBMC increased SR numbers significantly. Pirenzipine inhibited the CBMC-induced increase in SR numbers. On the basis of these findings, it is suggested that cholinergic agonists increase pineal SR numbers by acting through muscarinic M1 receptors. Hence muscarinic mechanisms may have a functional role in pineal physiology.

Acetylcholine↗

PubMed1987

Cell-wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii are (hydroxy)proline-specific proteases.

Two stage specific cell-wall lytic enzymes (autolysins) from different strains of the unicellular, biflagellated green alga Chlamydomonas reinhardtii were isolated and purified to homogeneity. Quantitative and specific photometric assays for biological activity were worked out to follow fractionation and to establish lytic specificity and kinetics. The autolysins were studied for enzymatic properties and screened for biological activity towards several wall components obtained by salt extractions of sporangia and zoospores from C. reinhardtii. The autolysins are proteolytic enzymes, fragmenting proline- or hydroxyproline-containing polypeptides in structures like connective tissue. They attack predominantly selected domains within the walls of zoosporangia or gametes. The sporangial autolysins are not only site- and strain-specific but also stage-specific, whereas the gamete autolysins lyse cell walls of gametes as well as those of sporangia and zoospores.

Cell Wall↗