Cor J. Grol

Norepinephrine release in the rat pineal gland: The input from the biological clock measured by in vivo microdialysis

Abstract: The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on‐line in vivo microdialysis. NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable to monitor the low levels of NE in the dialysates (12.5 fmol during nighttime, 3 fmol during daytime). To increase NE levels, the monoamine reuptake inhibitor cocaine was added to Ringers solution at concentrations of 10−6 and 10−5M. This resulted in increases of neurotransmitter output of 167 and 219%, respectively, but did not change the qualitative and/or quantitative outcome of other experiments. Perfusion with 10−6M tetrodotoxin for 1 h resulted in a decrease of the NE release by >80%, whereas perfusion with the α2‐receptor antagonist yohimbine caused a twofold increase. These results indicate that the NE release in the rat pineal was of neuronal origin and regulated by a negative feedback mechanism involving inhibitory presynaptic α2‐receptors. Long‐term (i.e., 16 h) measurements are described, showing the circadian properties of NE release. A pronounced rhythm is reported, showing extremely sharp transitions between low daytime and high nighttime values. Increases and decreases are reported to occur within the duration of collecting one sample (20 min). For comparison, the rhythm of melatonin release was also recorded. The on and off switches of the sympathetic input correlated well with the circadian rhythm of melatonin release and can thus be considered as the primary clock signal, inducing the nightly production of melatonin.

Microdialysis reveals dynamics of coupling between noradrenaline release and melatonin secretion in conscious rats

The coupling between noradrenergic innervation of the pineal gland and melatonin production was investigated. Previously, the development of a microdialysis technique was described which made it possible to study the noradrenaline (NA) input as well as the melatonin output with high time resolution. In the present study, we studied the effects of short-term changes in NA-release on melatonin secretion. A 1 min light pulse was applied around midnight and resulted in an immediate decrease of both NA and melatonin. While NA returned to basal levels in 40 min, melatonin did not reach the baseline within 2.5 h. This discrepancy in correlation between NA and melatonin indicates a rapid inactivation of N-acetyl-transferase (NAT), followed by a slow reactivation, possibly by de novo synthesis of NAT. During daytime, a perfusion with 60 mM potassium for 30 min, resulted in a rapid and short stimulation of NA release, which was not followed by an increase in melatonin production. This indicates that 30 min stimulation of NAT activation is not enough to increase the amount of melatonin produced. The combination of measuring NA input and melatonin output appears to be a valuable tool in studying the characteristics of pineal activity in great detail.

A telemetry study on the chronic effects of microdialysis probe implantation on the activity pattern and temperature rhythm of the rat

The present study describes the effects of implantation of microdialysis probes on temperature and activity rhythms of the rat, measured with a telemetry system. For comparison two widely used types of microdialysis probes were investigated, a transcerebral probe, inserted into the pineal gland and a set of two I-shaped concentric probes, implanted bilateral into the striatum. Starting from 5 days before the operation until 8 days after surgery, activity and temperature recordings were carried out continuously with the help of previously implanted transmitters. In separate experiments the effects of two different types of anaesthesia (chloralhydrate and Hypnorm) were determined. The results show that there is a pronounced effect of surgery on amplitude and rhythmicity of the temperature and activity patterns which is still detectable 6-7 days after operation. Few differences were noticed between the transverse probe and the I-shaped probes. Anaesthesia alone induced much smaller changes, most of which had disappeared within 2 or 3 days after the treatment. The duration of action of chloralhydrate is somewhat longer compared to Hypnorm. The conclusion is that when microdialysis is used in behavioural experiments, the effects of the surgical procedure should be taken into account. If these effects are serious, the use of previously implanted guide cannulae might be necessary.

The high affinity melatonin binding site probed with conformationally restricted ligands—II. Homology modeling of the receptor

We present the first 3-D model of the melatonin receptor based on the recently published amino acid sequence of the cloned melatonin receptor. The seven trans membrane helices were positioned using the helices found in the structure of Bacterio Rhodopsine. From the results of an indirect modeling study with six melatonergic agents, an alignment of these compounds was found directing towards common interaction points. These points are suggested to be the two serines in helix three and the histidine in helix five, forming hydrogen bonds with the amide function and the methoxy-oxygen in melatonin, respectively. The ligands were docked into these binding sites and the receptor-ligand complexes were energy minimized. Considering the position of the active and inactive ligands in the receptor and their respective occupied volumes, the structure-activity relationships are rationalized by the suggested model. This model can be of use as a pharmacological test model in molecular biological studies and as a basis to develop compounds being active as synchronizing circadian agents.

The high affinity melatonin binding site probed with conformationally restricted ligands- I. Pharmacophore and minireceptor models

The affinities of enantiomers of conformationally restricted melatonin analogues for the ML-1 and ML-2 putative melatonin receptor subtypes are reported. Most ligands exhibited reversed stereoselectivity when competing with 125I 2-iodomelatonin binding to chicken retinal (ML-1) and hamster brain (ML-2) membranes, further supporting the biochemical and pharmacological differences reported for these two sites. Based on the data for the ML-1 site and thorough conformational analyses of several ligands, two pharmacophore models were derived using the program APOLLO. The pharmacophoric elements included were putative receptor points from the amide NH, the amide CO, and the methoxy-O, together with the normal through the phenyl ring. The large drop in ML-1 affinity observed for 4-methoxy-2-acetamido-indan (6a) could not be explained from either of these models. Minireceptors were subsequently built around the two pharmacophores using Yak. Analysis of the resulting ligand-minireceptor interactions offered an explanation for the low affinity of 6a and allowed one of the pharmacophore models to be selected for use in future drug design.

Parasympathetic inhibition of pineal indole metabolism by prejunctional modulation of noradrenaline release

The role of the parasympathetic nervous system in rat pineal indole metabolism was investigated by transpineal in vivo microdialysis. On-line coupling to a high performance liquid chromatography system with fluorescence detection (HPLC-FD) allowed simultaneous analysis of three major indolic compounds from the pineal, i.e. serotonin, N-acetylserotonin and melatonin. Infusion of the muscarinic receptor agonists, carbachol and oxotremorine, during the dark period resulted in a marked decrease of melatonin release. This effect was suggested to be mediated by a decrease in N-acetyltransferase activity, since a similar decrease was seen in N-acetylserotonin release, while serotonin levels increased simultaneously. Nicotine did show a very slight effect on the three indoles under these circumstances. Neostigmine failed to influence pineal indole metabolism, indicating that the endogenous tonus of acetylcholine release is either absent or extremely low in the middle of the dark period. The involvement of sympathetic innervation in the muscarinic effects was investigated by measurement of noradrenaline release from the pineal by sensitive off-line HPLC-FD analysis of noradrenaline in the dialysates. Carbachol markedly decreased the noradrenaline input during the infusion. Noradrenaline release returned to baseline values immediately after infusion with carbachol. These data suggest that the in vivo inhibitory effect of muscarinic receptor agonists on pineal melatonin production is mediated by presynaptic muscarinic receptors, located on the sympathetic nerve endings. This prejunctional inhibition of noradrenaline release causes a reduced induction of N-acetyltransferase activity, resulting in decreased melatonin release.

Effect of non-catecholic 2-aminotetralin derivatives on dopamine metabolism in the rat striatum

SummaryThe concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in the striatum of rats after i.p. injection of dipropyl-2-aminotetralin and the four positional isomers of monohydroxy-dipropyl-2-aminotetralin. All compounds except 8-OH dipropylaminotetralin caused a decrease in DOPAC-and HVA-concentrations. In addition, 5-OH-dipropylaminotetralin produced a small elevation in DA concentrations. In contrast, 7-OH dipropylaminotetralin, in doses of 100 μmol/kg and more, decreased DA to 50% and initially increased HVA and DOPAC to about 200%, after which the concentrations of the metabolites fell to 30% or less. The 5-OH isomer was found to be the most potent compound, decreasing HVA concentrations to 70% at a dose of 0.14 μmol/kg. The potencies are compared to those of catechol-group containing DA-agonists such as apomorphine and N,N-dipropyl-5,6-dihydroxy-2-aminotetralin. In addition, a comparison is made with reported behavioural data. It is suggested that the more active N-alkylated 2-aminotetralins have a conformation which corresponds to that of the α rotamer of dopamine.

Effect of dihydroxy-2-aminotetralin derivatives on dopamine metabolism in the rat striatum

SummaryConcentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in the striatum of rats after i.p. injection of apomorphine, N,N-dipropyldopamine and a series of alkylated and/or esterified dopamine analogues of the dihydroxyaminotetralin type.All compounds tested caused a decrease in DOPAC- and HVA-concentrations. The N-alkylated derivatives had a rapid onset of action, showing a maximal HVA decrease after 15–45 min, after which time the metabolite concentrations slowly returned to control values. In addition, the dihydroxyaminotetralins, especially N,N-dipropylamino-5,6-dihydroxytetrahydronaphthalene (DiPr-5,6-ADTN), produced a rapid, short lasting elevation of DA concentrations. The esterified primary amines, dibenzoyl-5,6-and dibenzoyl-6,7-dihydroxyaminotetralin, had a delayed effect, causing a maximal HVA decrease after 4–6 h.DiPr-5,6-ADTN was found to be the most potent compound, with a maximal effect at a dose of 0.33 μmol/kg, it being 30 times more potent than apomorphine and DiPr-6,7-ADTN. The results corroborate reported behavioural data, and the relative potencies of the alkylated derivatives in this test system for dopaminergic activity are in agreement with those based on stereotyped behaviour.

Dopaminergic prodrugs: Brain concentrations and neurochemical effects of 5,6- and 6,7-ADTN after administration as dibenzoyl esters

Abstract Regional levels of the dopamine agonists 5,6- and 6,7-ADTN in rat brain were studied after administration of the parent compound and/or the dibenzoyl-ester derivative (a prodrug). A method for measuring 5,6- and 6,7-ADTN in the picomole range is described. The method is based on high performance liquid chromatography in conjunction with an electrochemical detector. ADTN levels in brain after administration of the dibenzoyl prodrug were about 5 times higher than when non-esterified ADTN was applied. The concentrations of the 5,6-isomer were 5–7 times higher than those of the 6,7-isomer. Constant brain levels of 5,6-ADTN (for at least 10 h) indicated that this isomer leaves the brain only with great difficulty. The susceptibility to metabolic degradation appears to play a crucial role in the in vivo activity of ADTN derivatives. An accumulation of 5,6-ADTN in DA-rich areas of the brain was observed after administration of the dibenzoyl derivative. By comparing the striatal levels of both ADTN isomers (after administration as dibenzoyl derivatives) with the data on their potency to decrease homovanillic acid concentrations in this brain area, we were able to quantitatively determine the in vivo activity of these isomers. 6,7-ADTN appeared to be about 12–14 times more potent than the 5,6-derivative, at the receptor site. These results strongly support the hypothesis that the active conformation of dopamine is closer to the β-rotamer than to the α-rotamer.

Conformational analysis of dopamine by the INDO molecular orbital method

The results of INDO calculations on dopamine are reported. A conformational energy map and an isodistance map for the key distances N—OH1, N—OH2 in dopamine as functions of the two main torsion angles τ1 and τ2 were constructed. In addition to the three known minima of dopamine corresponding to the trans and gauche forms, two new minima were found. The key distances of the rigid analogues of dopamine, apomorphine, isoapomorphine, 2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene and isoquinoline were plotted on the isodistance map of dopamine. By taking the corresponding τ values as coordinates on the energy map, conformations of dopamine, resembling the rigid analogues, could be found. When a conformation is close to a local minimum it is assumed that this conformation is energetically favourable. The possible relation between the energy minima and the biological action of dopamine is discussed. An explanation is suggested for the lack of dopaminergic activity of isoapomorphine.