Serotoninski sustav u trombocitima zdravih ljudi [ Platelet serotonin system in healthy human ]

Balija, Melita (2003) Serotoninski sustav u trombocitima zdravih ljudi [ Platelet serotonin system in healthy human ]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Serotonin is regarded as a master control neurotransmitter of complex neuronal communication. The brain serotonin system is involved in numerous physiological functions among which eating, sleep, sexual behaviour, circadian rhytmicity and neuroendocrine functions are the most prominent ones. The physiological role of high serotonin concentration in platelets has remained poorly understood. Serotonin was recently shown to play, in addition to its direct agonistic action, an important role as potent amplifier of platelet response. Blood platelets contain 5HT elements similar to neurons: two of them are in platelet cytoplasm: amine storage granules and mitochondrial MAO-B activity; others are linked to the platelet membrane: 5HT transporter and serotonin (5HT-2A) receptor. Platelet serotonin is stored in dense granules, analogously to vesicular 5HT in neurons, where is protected from MAO activity. However, in contrast to neurons, platelets do not synthesise 5HT and their amine content is a result of the transporter-mediated uptake from the surrounding blood plasma. Easy accessible platelet serotonin elements represent object of studies in neurolobiology and biological psychiatry as well as in various investigations addressing serotonin pathophysiology in general in contrast to far fewer studies aimed at the physiological aspects of platelet 5HT elements. This work has been aimed at the study of three platelet 5HT elements: platelet granular serotonin (PS), platelet serotonin transporter (5HTt) and platelet monoamineoxidase activity (MAO-B) in a population of 63 healthy human volunteers of both sexes. The first part of the investigation was dedicated to the introduction of methodologies enabling reliable measurements of the mentioned parameters. In the second part of this work we have investigated the main physiological characteristics of three platelet 5HT elements: frequency distribution of individual values, dependence on age, influence of sex, intra-individual stability over time and periodic oscillations, on a 63 healthy voluntary blood donor in the course of longer time period. Studies were performed on a population of healthy volunteers (blood donors) of both sexes: 10 females and 53 males aged between 21 and 67 years, over the period of 18 months. Blood sampling was routinely performed during the blood donor session. From each person a sample of 16 ml of venous blood was collected on 4 ml of ACD anticoagulant. After centrifuging blood in adapted plastic syringes PRP was quantitatively separated in aliquots of 1 ml. Platelet number and mean platelet volume, as well as measurement of 5HT transport kinetics was performed on the same day. The samples of platelet pellets (for measurement of serotonin level), and samples of PRP (for measurement of MAO-B kinetics) were frozen and stored at -20oC; the measurements were performed within two weeks. Platelet serotonin content was determined spectrophotofluorometrically. Deionized water was added to each platelet pellet and samples were sonicated. ZnSO4/NaOH and centrifugation precipitated proteins. Following deproteinisation of the ultrasonicated platelet sample, platelet serotonin was measured by orthophtaldialdehyde (OPT) – enhanced fluorometry at 345/485 nm. Platelet serotonin level was expressed per unit number of platelets (ng 5HT/109platelets) reflecting the amine concentration in platelets or per unit volume of whole blood (ng 5HT/ml) reflecting the amine level in the circulation. Platelet 5HT uptake was assayed by use of radioisotopic method. Platelets in Krebs-Ringer phosphate buffer (without CaCl2) were incubated with six concentrations of radioactively labelled (14C) serotonin. Uptake interval amounted to 60 seconds with sharp termination by the addition of ice-cooled buffer followed by immediate vacuum filtration. The determination of blank values was processed in the same manner but near 0oC. Results were expressed as Vmax in pmol of serotonin per 108 of platelets and Km in molar concentration of serotonin (µM). The kinetic product (Vmax/Km) was also calculated in each person. Activity of MAO-B was measured spectrophotofluorometrically using six concentrations of kynuramine as substrate. PRP was preincubated with buffer (Na-borate-HCl) and then incubated with six concentrations of substrate. The reaction was stopped by adding trichloroacetic acid, the mixture was centrifuged and formed 4-hydroxyquinoline (4-hQ) was measured fluorimetrically at 310 nm excitation and 362 nm emission. Standards and blanks were carried through the entire procedure. Km results were expressed as µM concentration of kynuramine. Vmax results were expressed as quantity of product 4-OH quinoline (nmol/108 platelets/60 min). The mean values of granular serotonin amounted to 537 ± 150,7 ng/109 platelets and the mean values of serotonin concentration in blood were 174 ± 63,4 /ml. The values of transporter kinetic parameters were: Vmax=138 ± 25,4 pmol/108/min and Km=0,400 ± 0,1 μM. The values of kinetic parameters of MAO-B activity were: Vmax=14,56 ± 4,9 nmol/108/h and Km=3,76 ± 1,1 μM. The studied parameters - granular serotonin content in platelets and serotonin level in blood, as well as kinetic parameters of serotonin transporter (Vmax and Km) and monoamineoxidase activity (Vmax and Km) demonstrated normal frequency distributions of individual values (with the bordeline values p=0,05 of Km distribution) of MAO-B. Regarding physiological characteristics - influences of age, sex and seasonal periodicity, significant changes were found in seasonal oscillation of platelet serotonin level sprig vs autumn (p<0,001) and autumn vs winter (p<0,01) and in MAO-B activity and transporter velocity. 5HT transporter velocity (Vmax) demonstrated verificant decrease with age (p<0,05) and higher values (Vmax/Km) in spring vs autumn (p<0,001) and in summer vs autumn (p<0,01). Velocity of MAO-B (Vmax) and Km demonstrated increase with age (p<0,05) and higher values (Vmax and Vmax/Km) in females (p<0,05) as well as significant seasonal oscillations Km in spring vs summer (p<0,001) and spring vs autumn (p<0,001) and oscillation Vmax/Km in spring vs summer (p<0,001), spring vs autumn (p<0,01) and spring vs winter (p<0,01). Kinetic parameter of serotonin transporter Vmax (maximal velocity) demonstrated high significant correlation with granular serotonin content (p<0,001) and with affinity of the transporter (p<0,001), but both platelet serotonin and kinetic parameters of serotonin transporter demonstrated no correlation with MAO-B kinetic parameters.

Abstract in Croatian

Serotonin pripada u glavne neurotransmitere koji sudjeluju u složenim neuronskim komunikacijama. Serotonin posreduje čitav niz centralnih i perifernih funkcija. Među ostalim, smatra se da je uključen u modulaciju brojnih fizioloških funkcija kao što su prehrana, spavanje, seksualno ponašanje, cirkadijani i neuroendokrini ritmovi i drugo. Trombocitni 5HT potječe iz krvne plazme jer trombocit ne posjeduje ključni enzim za sintezu 5HT - triptofan hidroksilazu te se pomoću prijenosnog proteina unosi se u unutrašnjost trombocita. Fiziološka uloga visokih koncentracija serotonina u trombocitima zdravih osoba još uvijek nije potpuno razjašnjena. Sam 5HT pripada u kategoriju slabih trombocitnih agonista ali je snažan pojačivač trombocitnog odgovora u prisutnosti drugih agonista. Elementi trombocitnog serotoninskog sustava gotovo su identični onima u serotonergičnim neuronima: serotoninski transmembranski prijenosnik (5HTt), serotoninski receptor (5HT-2A), monoaminoksidaza (MAO-B) i serotonin pohranjen u gustim granulama. Poremećaj serotonergičnih mehanizama veže se uz etiopatogenezu različitih duševnih i somatskih bolesti. Lako dostupni trombocitni serotoninski elementi predstavljaju objekt istraživanja u neurobiologiji i biološkoj psihijatriji kao i u različitim istraživanjima serotoninske patofiziologije. Međutim, vrlo rijetko se susreću radovi posvećeni fiziološkim aspektima trombocitnih 5HT elemenata. Ovaj rad smo posvetili fiziološkim istraživanjima tri elementa trombocitnog serotoninskog sustava u humanim trombocitima: koncentracije trombocitnog serotonina, aktivnosti serotoninskog prijenosnika na trombocitnoj membrani i aktivnosti trombocitne monoaminoksidaze. U prvom dijelu smo razradili metodološke pristupe koji omogućuju simultano mjerenje ta tri trombocitna serotoninska parametra. Nakon toga smo opisali glavne fiziološke osobine ta tri parametra u populaciji zdravih ispitanika: distribuciju frekvencija individualnih vrijednosti, utjecaj dobi, spola i godišnjih doba kroz vremensko razdoblje od 18 mjeseci. Na kraju smo istražili stupanj međusobne povezanosti (međuzavisnosti) navedenih parametara u fiziološkim uvjetima s ciljem dobivanja uvida u njihov stvarni međuodnos i s tim u vezi opravdanost korištenja pojma tzv. trombocitnog serotoninskog sustava. Ispitivanje je provedeno u 63 zdrava dobrovoljna darivatelja krvi starosti od 21 do 67 godina, oba spola (10 žena i 53 muškaraca), tijekom 18 uzastopnih mjeseci. Ispitanike smo podijelili u dvije ekstremne dobne skupine (mlađi od 30 i stariji od 50 godina). Uzorci krvi uzimani su od dobrovoljnih darivatelja krvi prilikom rutinskog davanja krvi. Od svakog ispitanika uzet je uzorak od 16 mL venozne krvi (+ 4 mL ACD antikoagulantne otopine). Iz uzorka pune krvi izdvojena je plazma bogata trombocitima (PRP) i podijeljena u alikvote za ispitivanje. Postupak mjerenja aktivnosti serotoninskog prijenosnika obavljen je unutar 2 sata nakon priprave PRP, a broj trombocita u PRP i punoj krvi određen je tijekom istog dana. Koncentracija trombocitnog serotonina mjerena je iz smrznutog trombocitnog taloga dobivenog centrifugiranjem PRP, a kinetika MAO-B iz smrznutih uzoraka plazme bogate trombocitima unutar 2 tjedna od spremanja uzoraka na –20oC. Koncentracija trombocitnog/cirkulatornog serotonina (5HT) mjerena je spektrofotofluorimetrijski. Trombocitni talog je homogeniziran ultrasonikacijom u deioniziranoj vodi. Proteini su precipitirani sa ZnSO4/NaOH, a sadržaj serotonina je kvantificiran primjenom OPT-fluorimetrijske metode. Standardi i prazne probe procesirane su istovjetno kao i uzorci. Rezultati su izraženi u ng 5HT po jediničnom broju trombocita (ng 5HT/109 trc) i na jedinični volumen krvi (ng 5HT/mL). Aktivnost trombocitnog serotoninskog prijenosnika određivana je radioizotopskom metodom uz primjenu radioaktivno obilježenog serotonina (14C-5HT). Trombociti resuspendirani u Krebs-Ringer fosfatnom puferu, bez CaCl2, inkubirani su u šest koncentracija 14C-5HT. Inkubacija je prekinuta dodatkom ledene fiziološke otopine i brzom vakuum filtracijom. Nakon ispiranja, radioaktivnost zaostala na filterima mjerena je beta scintilacijskim brojačem. Standardi i prazne probe procesirane su istovjetno kao i uzorci. Kinetički parametar Km izražen je kao molarna koncentracija 14C-5HT (µM), a maksimalna brzina (Vmax) u pmol 14C-5HT/108trombocita/minutu. Aktivnost trombocitne MAO-B određivana je iz PRP spektrofotofluorimetrijski uz kinuramin kao supstrat. Trombociti, preinkubirani u Na-borat-HCl puferu inkubirani su u šest koncentracija supstrata. Reakcija je prekinuta dodatkom trikloroctene kiseline i mješavina je centrifugirana. Intenzitet fluorescencije 4-hidroksikinolina (4-OH-kinolin) mjerio se na spektrofotofluorimetru. Standardi i prazne probe procesirane su istovjetno kao i uzorci. Kinetički parametar Km izražen je kao mM koncentracija supstrata. Aktivnost enzima (Vmax) izražena je kao količina produkta (4-OH-kinolin) nastala po jediničnom broju trombocita u jediničnom vremenu (nmol/108 trc/1h). Srednja vrijednost koncentracije trombocitnog serotonina iznosila je M=537±150,7 ng/109/trc, a srednja vrijednost koncentracije cirkulatornog serotonina M=174±63,4 ng/mL krvi. Vrijednosti kinetskih parametara serotoninskog prijenosnika bili su: Km=0,40±0,1 µM i Vmax=138±25,4 pmol/108trc/min. Vrijednosti kinetskih parametara aktivnosti MAO-B bili su: Km=3,76±1,1 μM i Vmax=14,56±4,9 nmol/108trc/h. Istraživani parametri – koncentracija trombocitnog/cirkulatornog srotonina i kinetski parametri Vmax i Km serotoninskog prijenosnika i aktivnosti monoaminoksidaze MAO-B pokazali su normalnu distribuciju frekvencija individualnih vrijednosti s graničnom vrijednosti Km za MAO-B (p=0,05). U odnosu na fiziološke karakteristike – utjecaj životne dobi, spola i godišnjeg doba, uočene su značajne varijacije koncentracije cirkulatornog serotonina u proljeće vs jesen (p<0,001) i jesen vs zima (p<0,01). Značajne promjene utvrđene su u brzini aktivnog unosa serotonina i u aktivnosti MAO-B. 5HT prijenosnik pokazao je značajan pad brzine (Vmax) u starijoj životnoj dobi (p<0,05) te porast učinkovitosti (Vmax/Km) u proljeće vs jesen (p<0,001) i ljeto vs jesen (p<0,01). Kinetski parametri MAO-B pokazali su značajan porast sa starijom životnom dobi (Vmax p<0,05; Km p<0,05) i značajno više vrijednosti Vmax (p<0,05) i Vmax/Km (p<0,05) u žena. Značajne sezonske varijacije Km kinetskog parametra MAO-B utvrđene su u proljeće vs ljeto (p<0,001) i proljeće vs jesen (p<0,001), a Vmax/Km proljeće vs ljeto (p<0,001), proljeće vs jesen (p<0,01) i proljeće vs zima (p<0,01). Analizom linearne korelacije utvrđena je statistički visoko značajna (p<0,0001) pozitivna povezanost koncentracije trombocitnog serotonina i maksimalne brzine (Vmax) unosa serotonina u trombocite te visoko značajna (p<0,0001) pozitivna povezanost maksimalne brzine (Vmax) unosa serotonina u trombocite i Michaelisove konstante (Km) kao mjere afiniteta trombocitnog serotoninskog prijenosnika. Korelacija između koncentracije trombocitnog serotonina ili kinetskih parametara trombocitnog serotoninskog prijenosnika s kinetskim parametrima trombocitne MAO-B nije utvrđena.

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