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Action of reserpine on subcellular 5-hydroxytryptamine organelles of blood platelets
Life Sciences Vol. 7, Part I, pp . 477-480, 1988, Printed in Great Britain.
Pergamon Press
ACTION OF RESERPINE ON SUBCELLULAR 5-HYDROXYTRYPTAMINE ORGANELLES OF BLOOD PLATELETS M . Da Prada, A . Pletscher, J .P . Tranzer and H . Knuchel Research Department, F . Hoffmann-La Roche & Co . Ltd ., Basle, Switzerland (Received 14 February 1988) IN blood platelets of various species, 5-hydroxytryptamine (SFiT) is stored to a great part in specific subcellular organelles which are highly osmiophilic, probably due to their 5HT content (1,2) . Recently, these organelles were isolated by density gradient centrifugation and shown to contain histamine and adeno-
sine triphosphate (ATP) besides 51FT (~,4) . Reserpine which liberatPS SFIT considerably decreases the number of these osmiophilic bodies in normal platelets (1,2,5-7) . It has not, however, been
inve,t .igated whether the organelles disappear in toto (e .g . by expulsi~n from the platelets or duc to intracellular destruction) or. whether empty" organelles, i .e . devoid of SHT, remain within the platelet, .
In this paper attempts were made to analyse the 5HT organelles of rabbits after pretreatment of the animals with reserpine . hx erim ental Rabbits of 2 - 3 kg were pretreated with 5 mg%kg reserpine i .p . prior to exsanguination . TFie platelets were isolated, homogenized by ultrasonication, differentially centrifuged and submitted
to density gradient centrifugation as indicated earlier (3) . ?he bottom layer previously shown to contain the 5HT organelles (3) was examined by electron microscopy . Furthermore, the following measurements mere carried out in this fraction : 5HT and histamine by spectropüotofluorimetric procedures (8,9), ATP with luciferinluciferase (10), and proteins colorimetrically (11) .
477
478
PLATELET 5HT
Vol . 7, No . 9
Results and Discussion After density gradient centrifugation of platelet homogenates from reserpinized rabbits a similar bottom layer was obtained as in the experiments with platelets from control animals . It consis
ted of a fine film attached to the wall of the centrifuge tube (3,4) . In contrast to the experiments with non-reserpinized rabbits, the bottom layer contained very little 5HT (about 0 .5 ;$ of controls) whereas histamine and ATP were only decreased to 34 and 63 9b respectively (Table) . The protein content of the bottom layer was in the same range as that in controls . TABLE Content of 5-Hydroxytryptamine ($HT), Histamine and Adenosine Triphosphate (ATP) in ymoles~mg Protein in the Bottom Layer consisting of Isolated jHT Organelles of Blood Platelets of Rabbits . 5 mg~kg reserpine were administered i .p . 16 hours before isolation of the platelets . Mean values and S .E . of 11 (controls) and 6 (reserpine) experiments respectively . Controls
Reserpine
ymoles 5HT
ymoles
z1 .6 + 1 .4
Histamine
8 .9 + 0 .~
ATP
8 .8 + 1 .1
0 .l _+ o .o +
,4~. o .j
3 .0 + 0 .6
3s
j .6 + 0 .9
63
On electron microscopic examinàtiun, the bottom layer consisted exclusively of vesicle-like structures . These were similar to those previously found in normal platelets (1,G), i .c" , they showed
a diameter of $00 - 2200 ~ and were surrounded by a Single memhra~ie" which appeared intact . The great majority of the ~-e~icles acre " empty, only a minority were partially or totall~~ filled with an usmiu-
philic material . In contrast, most of the vesicles from the plat~"lets of non-reserpinized animals contained an intensely usmiuphilic material which, as previously shown (1-4,7), probably consists of 5HT (Figure) .
These experiments indicate that the reserpine-induced liberation of 5HT from platelets in vivo is . not caused by- a destruction of 5HT organelles or by their in toto expulsion from the platelets .
Vol. 7, No. 9
PLATELET 5HT
479
FIG . Electron micrographs of the 5-hydroxytryptamine organelles of the bottom layer obtained by density gradient centrifugation of homogenates of rabbit platelets . Left : normal animals Right : animals pretreated with 5 mg/kg reserpine i .p . 16 hours prior to isolation of platelets Reserpine apparently liberates the remain intact within the platelets ted and can be isolated by density agreement with earlier findings on
5HT from the. organelles which after the amine has been deple-
gradient centrifugation . In whole platelets (12-14), reserpine affects the histamine and especially the ATP of the organelles less markedly than the 5HT . The $HT organelles of the platelets seem therefore to differ from the catecholamine granules of the adrenal medulla and from the $HT granules of the intestinal mucosa, since in these reserpine decreases the amines and ATP to about the same extent (15-1~) . Summary From platelets of reserpinised rabbits, a pure fraction of vesicle-like structures was obtained by density gradient centrifugation . The majority of these vesicles contained no osmiophilic substance, but their physical and morphological characteristics were otherwise similar to the previously described 5-hydroxy-
480
PLATELET 5 HT
Vol . 7, No. 9
tryptamine (5HT) organelles obtained from normal platelets . The vesicular fraction from reserpinized platelets contained much less
5HT than that from normal platelets, whereas histamine and especially adenosine triphosphate (ATP) were not as markedly decreased as 5HT.
References 1 . J .P . Tranzer, M . Da Prada and A . Pletscher, (1966) . J .P . Tranzer, A. Pletscher und M . Da Prada, 2. 24, C1o8 (1966) . 3 " M . Da Prada, A. Pletscher, J .P . Tranzer and 216, 1315 (1967) . 4 . M . Da Prada, A. Pletscher, J .P . Tranzer und physiol . Acta , in the press . 5 . M . Da Prada, J .P . Tranzer and A . Pletscher, Ther . 158 , 394 (1967) " 6 . J .P . Tranzer, M . Da Prada and A . Pletscher, cology , vol . 6, in the press .
Nature 212, 1574 Helv . physiol . Acta H . Knuchel, Nature H . Knuchel, Helv . J . Pharmacol . exp . Advances in Pharma -
7 . I .J . Bak, R . Hassler, B . May and E . Yestermann, Life Sci . s 6, 1133 (1967) . 8 . D .F . Bogdanski, A . Pletscher, B .B . Brodie and S . Udenfriend, J . Pharmacol . exp . Ther . ~, 82 (1956) . 9 . J .A . Huff, V.E . Davis and H . Brown, J . Lab . clin . Ned . 67, 1044 (1966) . 10 . H . Holmsen, I . Holmsen and A . Bernhardsen, Analyt . Biochem. 456 (1966) . 11 . O .H . Lowry, N .J . Rosebrough, A .L . Farr and R .J . Randall, J . biol . them . ~, z65 (1951) .
12 . A . Burkhalter, V .H . Cohn and P .A . Shore, Biochem. Pharmacol . 3z8 (1960) . 13 . G .V .R . Born, G .I .C . Ingram and R .S . Stacey, Brit . J . Pharmacol . ~, 62 (1958) . 14 . E . Yeber, U . Rose and Y . Ungar, Arch . int . Pharmacodyn . 166, 37 (1967) . 15 . N .A . Hillarp, Naturé 187, 1032 (1960) . 16 . H .J . Schümann, Arch . exp . Path . Pharmacol . 2~~, 237 (1958) " 17 . Y .H . Prusoff, Brit . J . Pharmacol . ~, 87 (1961) .
Pergamon Press
ACTION OF RESERPINE ON SUBCELLULAR 5-HYDROXYTRYPTAMINE ORGANELLES OF BLOOD PLATELETS M . Da Prada, A . Pletscher, J .P . Tranzer and H . Knuchel Research Department, F . Hoffmann-La Roche & Co . Ltd ., Basle, Switzerland (Received 14 February 1988) IN blood platelets of various species, 5-hydroxytryptamine (SFiT) is stored to a great part in specific subcellular organelles which are highly osmiophilic, probably due to their 5HT content (1,2) . Recently, these organelles were isolated by density gradient centrifugation and shown to contain histamine and adeno-
sine triphosphate (ATP) besides 51FT (~,4) . Reserpine which liberatPS SFIT considerably decreases the number of these osmiophilic bodies in normal platelets (1,2,5-7) . It has not, however, been
inve,t .igated whether the organelles disappear in toto (e .g . by expulsi~n from the platelets or duc to intracellular destruction) or. whether empty" organelles, i .e . devoid of SHT, remain within the platelet, .
In this paper attempts were made to analyse the 5HT organelles of rabbits after pretreatment of the animals with reserpine . hx erim ental Rabbits of 2 - 3 kg were pretreated with 5 mg%kg reserpine i .p . prior to exsanguination . TFie platelets were isolated, homogenized by ultrasonication, differentially centrifuged and submitted
to density gradient centrifugation as indicated earlier (3) . ?he bottom layer previously shown to contain the 5HT organelles (3) was examined by electron microscopy . Furthermore, the following measurements mere carried out in this fraction : 5HT and histamine by spectropüotofluorimetric procedures (8,9), ATP with luciferinluciferase (10), and proteins colorimetrically (11) .
477
478
PLATELET 5HT
Vol . 7, No . 9
Results and Discussion After density gradient centrifugation of platelet homogenates from reserpinized rabbits a similar bottom layer was obtained as in the experiments with platelets from control animals . It consis
ted of a fine film attached to the wall of the centrifuge tube (3,4) . In contrast to the experiments with non-reserpinized rabbits, the bottom layer contained very little 5HT (about 0 .5 ;$ of controls) whereas histamine and ATP were only decreased to 34 and 63 9b respectively (Table) . The protein content of the bottom layer was in the same range as that in controls . TABLE Content of 5-Hydroxytryptamine ($HT), Histamine and Adenosine Triphosphate (ATP) in ymoles~mg Protein in the Bottom Layer consisting of Isolated jHT Organelles of Blood Platelets of Rabbits . 5 mg~kg reserpine were administered i .p . 16 hours before isolation of the platelets . Mean values and S .E . of 11 (controls) and 6 (reserpine) experiments respectively . Controls
Reserpine
ymoles 5HT
ymoles
z1 .6 + 1 .4
Histamine
8 .9 + 0 .~
ATP
8 .8 + 1 .1
0 .l _+ o .o +
,4~. o .j
3 .0 + 0 .6
3s
j .6 + 0 .9
63
On electron microscopic examinàtiun, the bottom layer consisted exclusively of vesicle-like structures . These were similar to those previously found in normal platelets (1,G), i .c" , they showed
a diameter of $00 - 2200 ~ and were surrounded by a Single memhra~ie" which appeared intact . The great majority of the ~-e~icles acre " empty, only a minority were partially or totall~~ filled with an usmiu-
philic material . In contrast, most of the vesicles from the plat~"lets of non-reserpinized animals contained an intensely usmiuphilic material which, as previously shown (1-4,7), probably consists of 5HT (Figure) .
These experiments indicate that the reserpine-induced liberation of 5HT from platelets in vivo is . not caused by- a destruction of 5HT organelles or by their in toto expulsion from the platelets .
Vol. 7, No. 9
PLATELET 5HT
479
FIG . Electron micrographs of the 5-hydroxytryptamine organelles of the bottom layer obtained by density gradient centrifugation of homogenates of rabbit platelets . Left : normal animals Right : animals pretreated with 5 mg/kg reserpine i .p . 16 hours prior to isolation of platelets Reserpine apparently liberates the remain intact within the platelets ted and can be isolated by density agreement with earlier findings on
5HT from the. organelles which after the amine has been deple-
gradient centrifugation . In whole platelets (12-14), reserpine affects the histamine and especially the ATP of the organelles less markedly than the 5HT . The $HT organelles of the platelets seem therefore to differ from the catecholamine granules of the adrenal medulla and from the $HT granules of the intestinal mucosa, since in these reserpine decreases the amines and ATP to about the same extent (15-1~) . Summary From platelets of reserpinised rabbits, a pure fraction of vesicle-like structures was obtained by density gradient centrifugation . The majority of these vesicles contained no osmiophilic substance, but their physical and morphological characteristics were otherwise similar to the previously described 5-hydroxy-
480
PLATELET 5 HT
Vol . 7, No. 9
tryptamine (5HT) organelles obtained from normal platelets . The vesicular fraction from reserpinized platelets contained much less
5HT than that from normal platelets, whereas histamine and especially adenosine triphosphate (ATP) were not as markedly decreased as 5HT.
References 1 . J .P . Tranzer, M . Da Prada and A . Pletscher, (1966) . J .P . Tranzer, A. Pletscher und M . Da Prada, 2. 24, C1o8 (1966) . 3 " M . Da Prada, A. Pletscher, J .P . Tranzer and 216, 1315 (1967) . 4 . M . Da Prada, A. Pletscher, J .P . Tranzer und physiol . Acta , in the press . 5 . M . Da Prada, J .P . Tranzer and A . Pletscher, Ther . 158 , 394 (1967) " 6 . J .P . Tranzer, M . Da Prada and A . Pletscher, cology , vol . 6, in the press .
Nature 212, 1574 Helv . physiol . Acta H . Knuchel, Nature H . Knuchel, Helv . J . Pharmacol . exp . Advances in Pharma -
7 . I .J . Bak, R . Hassler, B . May and E . Yestermann, Life Sci . s 6, 1133 (1967) . 8 . D .F . Bogdanski, A . Pletscher, B .B . Brodie and S . Udenfriend, J . Pharmacol . exp . Ther . ~, 82 (1956) . 9 . J .A . Huff, V.E . Davis and H . Brown, J . Lab . clin . Ned . 67, 1044 (1966) . 10 . H . Holmsen, I . Holmsen and A . Bernhardsen, Analyt . Biochem. 456 (1966) . 11 . O .H . Lowry, N .J . Rosebrough, A .L . Farr and R .J . Randall, J . biol . them . ~, z65 (1951) .
12 . A . Burkhalter, V .H . Cohn and P .A . Shore, Biochem. Pharmacol . 3z8 (1960) . 13 . G .V .R . Born, G .I .C . Ingram and R .S . Stacey, Brit . J . Pharmacol . ~, 62 (1958) . 14 . E . Yeber, U . Rose and Y . Ungar, Arch . int . Pharmacodyn . 166, 37 (1967) . 15 . N .A . Hillarp, Naturé 187, 1032 (1960) . 16 . H .J . Schümann, Arch . exp . Path . Pharmacol . 2~~, 237 (1958) " 17 . Y .H . Prusoff, Brit . J . Pharmacol . ~, 87 (1961) .