Platelet 5-Hydroxytryptamine Storage in the Carcinoid Syndrome An Electron Microscopic Autoradiographic Study JON C. LEWIS, PH.D., AND CHARLES G. MOERTEL, M.D.
THE UPTAKE of 5-hydroxytryptamine by blood platelets has been studied extensively during recent years, and data have accumulated to support the hypothesis that in mammalian platelets this amine is actively taken up and stored exclusively in specific cytoplasmic organelles. 3 - 41013 The ultrastructure of these organelles, referred to as "dense bodies," has been well described for platelets from nonhuman species, 414 and evidence suggests that similar storage sites exist in human platelets. 4 1 5 1 6 The evidence for these structures in normal human platelets, however, is inconclusive, because electron microscopic demonstration of "dense bodies" is dependent on the presence of divalent cations, principally calcium, in the fixation medium. 8 1 5 1 6 Received August 9, 1976; received revised manuscript June 6, 1977; accepted for publication June 21, 1977. Address reprint requests to Dr. Lewis: Department of Pathology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103.
Departments of Pathology, Bowman Gray School of Medicine, Winston-Salem, North Carolina, and Mayo Clinic and Mayo Foundation, Rochester, Minnesota
Recently, Minter and Crawford11 found more than one subcellular storage site for serotonin in mammalian platelets. Most important, however, these authors found that, in pig platelets, 80% of the cellular 5hydroxytryptamine was associated with an extragranular compartment. Because in the platelet-release reaction newly absorbed serotonin behaves essentially the same as endogenous 5-hydroxytryptamine, 6 a more precise identification of storage sites for newly absorbed serotonin is intrinsic to a complete understanding of human platelet function. Previously, we reported finding elevated numbers of dense bodies in human platelets from patients with the carcinoid syndrome. 7 As demonstrated through the use of dichromate staining7 or reserpine treatment, 9 these dense bodies apparently are storage sites for biogenic amines. In the present report we describe the results of an electron microscopic autoradiographic study of newly absorbed serotonin in platelets from normal donors and patients with the carcinoid syndrome. Our results support the hypothesis of two or more storage sites of 5-hydroxytryptamine in mammalian platelets. Materials and Methods Human blood obtained from informed volunteers was collected by venipuncture, anticoagulated with heparin (10 units/ml), and centrifuged for 10 minutes at 100 x g to prepare platelet-rich plasma. The clinical characteristics of the patients with carcinoid syndrome included in these studies have been described in detail elsewhere. 7 Briefly, platelets used in the autoradiographic studies were from three of these patients who had histologically confirmed metastatic tumors with primary tumors in the ileum. Elevated urinary levels of 5-hydroxyindoleacetic acid (5-
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Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Lewis, Jon C , and Moertel, Charles G.: Platelet 5-hydroxytryptamine storage in the carcinoid syndrome. An electron microscopic autoradiographic study. Am J Clin Pathol 70: 628-631, 1978. The uptake and storage of 5-hydroxytryptamine by human platelets from normal donors and patients with the carcinoid syndrome are reported. When platelets were incubated in the presence of 3H-5-hydroxytryptamine (50 ftM), an initial phase of rapid uptake was observed until 5-hydroxytryptamine levels of 1-2 /xg/109 cells were reached. Further accumulation occurred more slowly. Electron microscopic autoradiography of cells after 5-hydroxytryptamine uptake for 60 minutes revealed that in platelets from normal donors most of the label was localized in membranes (52%), a granules (27%), and cytoplasm (4%). Dense bodies were associated with 18% of the silver grains. Similar membrane (50%) and a-granule (27%) labeling was found with platelets from patients with carcinoid syndrome. However, more cytoplasmic labeling (10%) and less dense-body labeling (11%) were found with these cells. The results support a hypothesis of two or more sites for storage of 5-hydroxytryptamine in mammalian platelets and suggest that minimal exchange between cytoplasmic and dense-body amines occurs in carcinoid platelets. (Key words: Carcinoid platelets; 5-Hydroxytryptamine uptake; Mammalian platelets; Serotonin storage.)
Vol. 70. No. 4
PLATELET 5-HT STORAGE IN CARCINOID SYNDROME
Results Comparison of 5-hydroxytryptamine uptake by platelets from normal donors and patients with carcinoid syndrome shows that an initial period of rapid uptake is followed by a prolonged period during which cellular 5-hydroxytryptamine accumulates at a less rapid but steady rate (Fig. 1). In both populations of platelets, the rapid initial phase continues until 5-HT levels of 1-2 /xg/lO9 cells have been reached, after which further uptake is slower in platelets from the patients than in platelets from normal donors. These results are consistent with findings in a study reported by Crawford.2 When platelets incubated in this manner were prepared for autoradiography, silver grains were found associated with external membranes and membranes of the open-channel system, a granules, and other cytoplasmic structures, including mitochondria and phagocytic debris vacuoles (Fig. 2). Few background grains were observed on unlabeled control sections, and nonspecific silver grains (not * New England Nuclear, Boston, Mass. t Sigma Chemical Co., St. Louis, Mo. t Beckman, Palo Alto, Cal. § Ilford Ltd, Essex, England. 1 Philips EM-201 C, Mahwah, N. J.
-Si •v.
•v.
•v.
120 150 180 210 240
Incubation time (minutes) FIG. I. Platelet 5-hydroxytryptamine content in normal (open circles) and carcinoid (crosses) platelets after incubation in plasma containing 50 IJLM 5-hydroxytryptamine.
associated with cellular structures) accounted for less than 5% of the label in the experimental sections. More than 100 leukocyte sections were observed in the autoradiographic experiments but no silver grains were localized to these cells. The distributions of silver grains in normal and carcinoid platelets after uptake of [3H]5-HT are shown in Table 1. Membranes and a granules in both platelet groups were most frequently labeled, with membranes alone having 50% of the bound 5-hydroxytryptamine. Unexpectedly, although more than 50% of the carcinoid platelets had identifiable dense bodies (Fig. 2), only 11% of the silver grains was associated with dense bodies (Table 1). This can be compared with 18% dense-body-associated label in platelets from normal donors. Discussion After studying rabbit platelets, Pletscher and associates12 postulated that two mechanisms were involved in 5-hydroxytryptamine uptake by these cells. Transport at normal physiologic levels of 5-hydroxytryptamine apparently occurs in nonsaturated platelets through an energy-dependent "active" mechanism. Conversely, when storage sites linked to the "active" mechanism are saturated or are under conditions of unusually high levels of 5-hydroxytryptamine (>5 /LAg/ml), further transport of 5-hydroxytryptamine apparently occurs through passive diffusion. This hypothesis has been substantiated by Drummond and Gordon,5 who identified three sites, all with different affinities for 5-hydroxytryptamine, in the platelets of the rat.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
HIAA), as well as clinical manifestations such as flushing and diarrhea, were common manifestations. For serotonin uptake, 2-[:,H]-N-5-hydroxytryptamine binoxylate* was diluted with 5-hydroxytryptamine creatinine sulfatet in saline solution and was then added to the platelet-rich plasma so that the final concentration of serotonin was 50 /XM, with an activity of 0.1 ixCi/m\. Cells were then incubated at 37 C for 5, 30, 75, 120, or 240 minutes, fixed for 30 minutes in 2.5% glutaraldehyde buffered to pH 7.2 with 0.1 M phosphate, and washed three times in the same buffer to remove unbound label. The radioactivity was determined by counting 20% of the cell suspension in a scintillation counter.i In all experiments, platelet-rich plasma was diluted with platelet-poor plasma prepared from the same donor to give a final platelet count of 200,000/mm:'. For autoradiographic studies, cells were incubated in serotonin for 90 minutes and subsequently fixed as previously described above, postfixed in 1% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in epoxy resin (Epon 812). Thin sections, collected on carbon-stabilized, Formvar-coated, copper grids, were coated with Ilford L-4 emulsion§ and stored at 4 C for as long as four months. Grids were developed at periodic intervals in Microdol-X, stained with lead citrate and uranyl acetate, and viewed in an electron microscope.11
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THE UPTAKE of 5-hydroxytryptamine by blood platelets has been studied extensively during recent years, and data have accumulated to support the hypothesis that in mammalian platelets this amine is actively taken up and stored exclusively in specific cytoplasmic organelles. 3 - 41013 The ultrastructure of these organelles, referred to as "dense bodies," has been well described for platelets from nonhuman species, 414 and evidence suggests that similar storage sites exist in human platelets. 4 1 5 1 6 The evidence for these structures in normal human platelets, however, is inconclusive, because electron microscopic demonstration of "dense bodies" is dependent on the presence of divalent cations, principally calcium, in the fixation medium. 8 1 5 1 6 Received August 9, 1976; received revised manuscript June 6, 1977; accepted for publication June 21, 1977. Address reprint requests to Dr. Lewis: Department of Pathology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103.
Departments of Pathology, Bowman Gray School of Medicine, Winston-Salem, North Carolina, and Mayo Clinic and Mayo Foundation, Rochester, Minnesota
Recently, Minter and Crawford11 found more than one subcellular storage site for serotonin in mammalian platelets. Most important, however, these authors found that, in pig platelets, 80% of the cellular 5hydroxytryptamine was associated with an extragranular compartment. Because in the platelet-release reaction newly absorbed serotonin behaves essentially the same as endogenous 5-hydroxytryptamine, 6 a more precise identification of storage sites for newly absorbed serotonin is intrinsic to a complete understanding of human platelet function. Previously, we reported finding elevated numbers of dense bodies in human platelets from patients with the carcinoid syndrome. 7 As demonstrated through the use of dichromate staining7 or reserpine treatment, 9 these dense bodies apparently are storage sites for biogenic amines. In the present report we describe the results of an electron microscopic autoradiographic study of newly absorbed serotonin in platelets from normal donors and patients with the carcinoid syndrome. Our results support the hypothesis of two or more storage sites of 5-hydroxytryptamine in mammalian platelets. Materials and Methods Human blood obtained from informed volunteers was collected by venipuncture, anticoagulated with heparin (10 units/ml), and centrifuged for 10 minutes at 100 x g to prepare platelet-rich plasma. The clinical characteristics of the patients with carcinoid syndrome included in these studies have been described in detail elsewhere. 7 Briefly, platelets used in the autoradiographic studies were from three of these patients who had histologically confirmed metastatic tumors with primary tumors in the ileum. Elevated urinary levels of 5-hydroxyindoleacetic acid (5-
0002-9173/78/1000/0628 $00.60 © American Society of Clinical Pathologists
628
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Lewis, Jon C , and Moertel, Charles G.: Platelet 5-hydroxytryptamine storage in the carcinoid syndrome. An electron microscopic autoradiographic study. Am J Clin Pathol 70: 628-631, 1978. The uptake and storage of 5-hydroxytryptamine by human platelets from normal donors and patients with the carcinoid syndrome are reported. When platelets were incubated in the presence of 3H-5-hydroxytryptamine (50 ftM), an initial phase of rapid uptake was observed until 5-hydroxytryptamine levels of 1-2 /xg/109 cells were reached. Further accumulation occurred more slowly. Electron microscopic autoradiography of cells after 5-hydroxytryptamine uptake for 60 minutes revealed that in platelets from normal donors most of the label was localized in membranes (52%), a granules (27%), and cytoplasm (4%). Dense bodies were associated with 18% of the silver grains. Similar membrane (50%) and a-granule (27%) labeling was found with platelets from patients with carcinoid syndrome. However, more cytoplasmic labeling (10%) and less dense-body labeling (11%) were found with these cells. The results support a hypothesis of two or more sites for storage of 5-hydroxytryptamine in mammalian platelets and suggest that minimal exchange between cytoplasmic and dense-body amines occurs in carcinoid platelets. (Key words: Carcinoid platelets; 5-Hydroxytryptamine uptake; Mammalian platelets; Serotonin storage.)
Vol. 70. No. 4
PLATELET 5-HT STORAGE IN CARCINOID SYNDROME
Results Comparison of 5-hydroxytryptamine uptake by platelets from normal donors and patients with carcinoid syndrome shows that an initial period of rapid uptake is followed by a prolonged period during which cellular 5-hydroxytryptamine accumulates at a less rapid but steady rate (Fig. 1). In both populations of platelets, the rapid initial phase continues until 5-HT levels of 1-2 /xg/lO9 cells have been reached, after which further uptake is slower in platelets from the patients than in platelets from normal donors. These results are consistent with findings in a study reported by Crawford.2 When platelets incubated in this manner were prepared for autoradiography, silver grains were found associated with external membranes and membranes of the open-channel system, a granules, and other cytoplasmic structures, including mitochondria and phagocytic debris vacuoles (Fig. 2). Few background grains were observed on unlabeled control sections, and nonspecific silver grains (not * New England Nuclear, Boston, Mass. t Sigma Chemical Co., St. Louis, Mo. t Beckman, Palo Alto, Cal. § Ilford Ltd, Essex, England. 1 Philips EM-201 C, Mahwah, N. J.
-Si •v.
•v.
•v.
120 150 180 210 240
Incubation time (minutes) FIG. I. Platelet 5-hydroxytryptamine content in normal (open circles) and carcinoid (crosses) platelets after incubation in plasma containing 50 IJLM 5-hydroxytryptamine.
associated with cellular structures) accounted for less than 5% of the label in the experimental sections. More than 100 leukocyte sections were observed in the autoradiographic experiments but no silver grains were localized to these cells. The distributions of silver grains in normal and carcinoid platelets after uptake of [3H]5-HT are shown in Table 1. Membranes and a granules in both platelet groups were most frequently labeled, with membranes alone having 50% of the bound 5-hydroxytryptamine. Unexpectedly, although more than 50% of the carcinoid platelets had identifiable dense bodies (Fig. 2), only 11% of the silver grains was associated with dense bodies (Table 1). This can be compared with 18% dense-body-associated label in platelets from normal donors. Discussion After studying rabbit platelets, Pletscher and associates12 postulated that two mechanisms were involved in 5-hydroxytryptamine uptake by these cells. Transport at normal physiologic levels of 5-hydroxytryptamine apparently occurs in nonsaturated platelets through an energy-dependent "active" mechanism. Conversely, when storage sites linked to the "active" mechanism are saturated or are under conditions of unusually high levels of 5-hydroxytryptamine (>5 /LAg/ml), further transport of 5-hydroxytryptamine apparently occurs through passive diffusion. This hypothesis has been substantiated by Drummond and Gordon,5 who identified three sites, all with different affinities for 5-hydroxytryptamine, in the platelets of the rat.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
HIAA), as well as clinical manifestations such as flushing and diarrhea, were common manifestations. For serotonin uptake, 2-[:,H]-N-5-hydroxytryptamine binoxylate* was diluted with 5-hydroxytryptamine creatinine sulfatet in saline solution and was then added to the platelet-rich plasma so that the final concentration of serotonin was 50 /XM, with an activity of 0.1 ixCi/m\. Cells were then incubated at 37 C for 5, 30, 75, 120, or 240 minutes, fixed for 30 minutes in 2.5% glutaraldehyde buffered to pH 7.2 with 0.1 M phosphate, and washed three times in the same buffer to remove unbound label. The radioactivity was determined by counting 20% of the cell suspension in a scintillation counter.i In all experiments, platelet-rich plasma was diluted with platelet-poor plasma prepared from the same donor to give a final platelet count of 200,000/mm:'. For autoradiographic studies, cells were incubated in serotonin for 90 minutes and subsequently fixed as previously described above, postfixed in 1% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in epoxy resin (Epon 812). Thin sections, collected on carbon-stabilized, Formvar-coated, copper grids, were coated with Ilford L-4 emulsion§ and stored at 4 C for as long as four months. Grids were developed at periodic intervals in Microdol-X, stained with lead citrate and uranyl acetate, and viewed in an electron microscope.11
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LEWIS AND MOERTEL
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