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Mechanism of anemia associated with autonomic dysfunction in rats
Autonomic Neuroscience: Basic and Clinical 82 (2000) 123–129 www.elsevier.com / locate / autneu
Mechanism of anemia associated with autonomic dysfunction in rats a b, a c c Konen Obayashi , Yukio Ando *, Hisayasu Terazaki , Taro Yamashita , Masaaki Nakamura , Moritaka Suga a , Makoto Uchino c , Masayuki Ando a a
First Department of Internal Medicine, Kumamoto University School of Medicine, 1 -1 -1 Honjo, Kumamoto 860 -0811, Japan b Department of Laboratory Medicine, Kumamoto University School of Medicine, 1 -1 -1 Honjo, Kumamoto 860 -0811, Japan c Department of Neurology, Kumamoto University School of Medicine, 1 -1 -1 Honjo, Kumamoto 860 -0811, Japan Received 20 December 1999; received in revised form 23 February 2000; accepted 23 February 2000
Abstract The aim of this study was to elucidate the mechanism of anemia associated with autonomic dysfunction in rats. Using 6hydroxydopamine (6-OHDA)-treated sympathectomized rats, changes in systolic blood pressure, plasma catecholamine levels, hemograms, erythropoietin (EPO) secretion, and b-adrenergic receptors on erythrocytes were monitored, and compared with desipramine- and 6-OHDA-treated, and control rats. In 6-OHDA-treated rats, systolic blood pressure and plasma catecholamine levels significantly decreased from 7 days after 6-OHDA administration, returning to the control values on day 28. Hemoglobin (Hb), hematocrit (Hct) and red blood cell (RBC) levels significantly decreased from day 14 to day 28, and reached normal values after day 35, but neither corpuscular constants nor white blood cell (WBC) levels changed after anemia occurred. Administration of desipramine 1 day before 6-OHDA injection prevented anemia. EPO levels did not elevate, even after bloodletting to load anemia, and the EPO circadian rhythm was irregular in 6-OHDA-treated rats. b-adrenergic receptors measured using 125 I-cyanopindolol (CYP) significantly decreased from day 7 to day 28, and reached normal values after day 35. These results suggest that irregular EPO secretion via disordered autonomic nerves may induce anemia in patients with autonomic disorders. 2000 Elsevier Science B.V. All rights reserved. Keywords: Anemia; Autonomic dysfunction; 6-OHDA; Erythropoietin; b-adrenergic receptors
1. Introduction Anemia is induced by various factors and pathological conditions, such as lowered red blood cell (RBC) survival time (Erslev, 1970; McGonigle et al., 1984; Eschbach and Adamson, 1985), hemolysis (Shaw, 1967; Eschbach and Adamson, 1985), hemorrhage, iron deficiency, retained toxic substances or uremic inhibitors that interfere with erythroid marrow function (Eschbach and Adamson, 1985) as well as inadequate secretion of erythropoietin (EPO) in the kidneys (Adamson et al., 1968; McGonigle et al.,
Abbreviations: FAP, familial amyloidotic polyneuropathy; ATTR, amyloidogenic transthyretin; Val, valine; Met, methionine; 6-OHDA, 6-hydroxydopamine; Hb, hemoglobin; Hct, hematocrit; RBC, red blood cell; WBC, white blood cell; EPO, erythropoietin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CYP, cyanopindolol *Corresponding author. Tel.: 181-963-735-692; fax: 181-963-735692. E-mail address: [email protected] (Y. Ando)
1984). Owing to the progress of biochemical and molecular genetic methods, the mechanism of several types of anemia has been explained, though many remain to be elucidated. It has been well documented that normocytic and normochromic anemia is sometimes associated with autonomic dysfunction (Asahara et al., 1993; Biaggioni et al., 1994; Ando et al., 1996): we recently reported that anemia was recognized in patients with familial amyloidotic polyneuropathy (FAP) amyloidogenic transthyretin Valine 30 Methionine (ATTR Val 30 Met), pandysautonomia and Shy–Drager syndrome, which are all noted for their severe autonomic dysfunction (Asahara et al., 1993; Ando et al., 1996). Among these three diseases, FAP ATTR Val30 Met shows the most severe autonomic dysfunction, such as orthostatic hypotension, diarrhea, pupillary disorder, decreased sweating, and dry eyes (Ando et al., 1993; Biaggioni et al., 1994), while exhibiting the most severe anemia correlated with the progression of autonomic dysfunction (Asahara et al., 1993; Biaggioni et al., 1994; Ando et al., 1996).
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Since autonomic nerves dominate the kidneys, bone marrow and surrounding tissues, erythropoiesis may be modified by disordered autonomic nerves (Ganong, 1973; Bulloch, 1985). Takaku et al. reported that the reticulocyte response to acute bloodletting was significantly diminished in rats when their kidneys were functionally denerved (Takaku et al., 1961), and there are several reports describing the growth of erythroid progenitor cells being enhanced in the presence of b-adrenergic stimulants in in vitro studies (Brown and Adamson, 1977; Prazala et al., 1977; Beckman and Fisher, 1979; Dresch et al., 1981; Mladenovic and Adamson, 1984). Moreover, Bazan reported the possible relationship between nervous system and hematopoiesis (Bazan, 1991). These results suggest that there is some erythropoietic regulation via the autonomic nervous system. However, the mechanism causing anemia associated with autonomic dysfunction is not well explained. It is also well known that 6-hydroxydopamine (6OHDA) is a useful neurotoxic agent which can reversibly impair the sympathetic nerve terminal (Thoenen and Tranzer, 1968). When it is injected intravenously or intraperitoneally, it accumulates in the peripheral sympathetic nerve terminal and selectively destroys the sympathetic nerves. Its toxic effects directly result from its ability to generate free radical species, and from covalent bonding of quinone oxidation product (Kostrzewa and Jacobowitz, 1974). With this chemical agent, we can create pure sympathectomized rats, which are reversible by the administration of desipramine, a competitive inhibitor of 6-OHDA (Tsao et al., 1996). Moreover, since the neurotoxic effect of 6-OHDA is reversible, we can follow up not only the chemically sympathectomized stage, but also the recovering stage of autonomic dysfunction using 6-OHDAtreated rats. The aim of this study was to elucidate the mechanism of anemia associated with autonomic dysfunction. In 6OHDA-treated rats, the following experiments were performed. (1) To examine the changes in physiological conditions, systolic blood pressure was monitored. (2) To examine the effect of 6-OHDA on catecholamine production in the tissues, plasma epinephrine and norepinephrine levels were measured. (3) To investigate the effect of 6-OHDA on hematopoiesis, changes in hemograms were compared between 6-OHDA-treated and control rats. (4) To investigate the response of EPO to the anemia, changes in serum EPO levels were measured in 6-OHDA-treated rats after bloodletting and compared with those in control rats. (5) To analyze the circadian rhythm of EPO secretion, serum EPO levels were monitored in 6-OHDA-treated rats four times a day and compared with those in control rats. (6) To determine the changes in b-adrenergic receptors on the surface of erythrocytes during anemia, the binding of cyanopindolol (CYP) to erythrocytes was examined in 6-OHDA-treated rats.
2. Materials and methods
2.1. Chemicals 6-OHDA hydrobromide and desipramine were purchased from Sigma Chemical (St. Louis, USA). 125 I-CYP reagent (2,200 Ci / mmol) was obtained from New England Nuclear (Boston, MA, USA). All chemicals used in this study were of analytical grade.
2.2. In vivo experiments To omit the androgenic effect on the secretion of EPO, 30 female Wistar rats (7-week-old, 150–200 g) were used in this study. These rats were fed a laboratory pellet diet and water, and were maintained under the same conditions. 6-OHDA was dissolved in saline containing 0.1% ascorbic acid immediately prior to injection. Fifteen rats were chemically sympathectomized via intravenous administration of 0.1 ml of 6-OHDA solution (containing 100 mg / kg of 6-OHDA). Another ten rats were administrated an equivalent volume of a vehicle (0.1 ml, iv) following the same procedure. Five of the 6-OHDA-treated rats were simultaneously injected with 0.1 ml of desipramine in saline (10 mg / kg) 1 day before 6-OHDA injection (Thoenen and Tranzer, 1968).
2.3. Systolic blood pressure Systolic blood pressure was monitored in fifteen 6OHDA injected rats at the tail using a blood pressure monitor, model PS-200S (Riken, Tokyo, Japan) from day 0 to 42.
2.4. Measurement of plasma epinephrine and norepinephrine levels in the 6 -OHDA-treated rats’ serum Three hundred microliters of plasma samples from 6OHDA-treated, 6-OHDA and desipramine-treated rats, and control rats were mixed with 300 ml of 6% perchloric acid solution in a vortex-mixer for deproteination. After adding 200 ml of 1.5 mol / l sodium acetate, the mixture was stirred and centrifuged (3000 rpm) at 28C for 20 min. The clear supernatant was applied to the autosampler of a high performance liquid chromatography (HPLC) analyzer (Tosoh, Tokyo, Japan; Eurogenetics, Tessenderlo, Belgium; Tosoh Medics, Emeryville, CA, USA) (Yoshimura et al., 1993).
2.5. Hemograms After collecting 0.2 ml of blood samples, hemoglobin (Hb), hematocrit (Hct), red blood cell (RBC) and white blood cell (WBC) levels were measured in five 6-OHDAtreated rats, five desipramine and 6-OHDA-treated rats,
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and five control rats on day 0, 3, 7, 14, 21, 28, 35 and 42 after treatment. In addition, we calculated the corpuscular constants [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)] from this data on day 28.
2.6. Serum EPO assay Serum EPO levels were measured in five 6-OHDAtreated rats and five control rats after 4 ml of bloodletting on day 7. The circadian rhythm of EPO secretion was determined in five 6-OHDA-treated rats and compared with that in five control rats. The blood samples were taken from the rats at 1: 00, 7: 00, 13: 00, and 19: 00. The EPO levels were measured using an EPO-specific ELISA kit (Kyokuto, Osaka, Japan).
2.7. b -adrenergic receptors on erythrocytes The number of b-adrenergic receptors was determined in the blood samples in five 6-OHDA injected rats and five control rats on day 0, 3, 7, 14, 21, 28, 35 and 42 after treatment. Binding of 125 I-CYP to erythrocytes was determined by incubating 5310 5 cells with 0.02 mCi of 125 I-CYP in 150 mM sodium chloride, 1 mM ascorbic acid, and 20 mM Tris, pH 7.4, for 90 min at 308C. Receptor density was determined by incubating the erythrocytes with at least five different concentrations of 125 ICYP, ranging from 25 to 200 pM. Non-specific binding was determined by measuring the amount of 125 I-CYP bound to the cells in the presence of 6 mM DL-propranolol. The experiments were terminated by dilution with 5 ml of ice-cold incubation buffer followed by vacuum filtration through Whatman GF / C filters. The sample tubes were rinsed with an additional 5 ml of the cold buffer and the filters were washed with an additional 30 ml of the icecold incubation buffer. The filters were packed into sample tubes and counted in a gamma counter, model ARC-361 (Aloka, Tokyo, Japan). The count per min (cpm) reflecting the real receptor density was corrected by deleting the non-specific binding values as described above.
2.8. Statistical analysis All the data were expressed as mean6S.D. The significance of the data was determined by unpaired Student’s t-test. P values less than 0.05 were considered significant.
3. Results
3.1. Change in systolic blood pressure after 6 -OHDA treatment To investigate changes in physiological conditions after
Fig. 1. Changes in systolic blood pressure in 6-OHDA-treated rats. Systolic blood pressure was monitored in the tail as described previously. The statistical significance was compared before and after administration of 6-OHDA. Fifteen rats were used for these experiments. * P,0.05.
6-OHDA injection, systolic blood pressure was monitored as demonstrated in Fig. 1. A significant decrease in blood pressure was observed from day 4 to 21 after 6-OHDA administration (P,0.05), and reached a normal value after day 28.
3.2. Measurement of plasma epinephrine and norepinephrine levels in the 6 -OHDA-treated rats’ serum To examine the effect of 6-OHDA on catecholamine production in the tissues, plasma epinephrine and norepinephrine levels were measured. As demonstrated in Fig. 2, both plasma epinephrine and norepinephrine levels significantly decreased in 6-OHDA-treated rats on day 7 after 6-OHDA treatment, returning to the control values on day 28. Desipramine injection effectively prevented the decrease in catecholamine levels.
3.3. Changes in hemograms To elucidate the effect of 6-OHDA on hematopoiesis, Hb, Hct, RBC, and WBC levels were measured in 6OHDA-treated rats and compared with those in control rats. Hb, Hct and RBC levels significantly decreased from day 14 to 28 (P,0.05), and reached the normal values after day 35 in 6-OHDA-treated rats. Desipramine intravenously administered 1 day before the 6-OHDA in-
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Fig. 2. Measurement of plasma epinephrine (A) and norepinephrine (B) levels in 6-OHDA-treated rats’ serum. Plasma epinephrine and norepinephrine levels were measured in 6-OHDA-treated, 6-OHDA- and desipramine-treated, and control rats. h: control rats, j: 6-OHDA-treated rats, and : 6-OHDA and desipramine-treated rats. Five rats from each group were used for these experiments. * P,0.05.
jection effectively prevented the occurrence of anemia (Fig. 3 A-C). Neither MCV, MCH, MCHC nor WBC levels prominently changed, even after anemia occurred in 6OHDA injected rats (Table 1, Fig. 4).
3.4. Changes in EPO secretion during anemia To check the EPO response in 6-OHDA-induced anemia, serum EPO levels were measured in 6-OHDA-
Fig. 3. Change in the hemograms in 6-OHDA-treated rats. Hb (A), Hct (B), and RBC (C) levels were measured in 6-OHDA-treated, 6-OHDA and desipramine-treated, and control rats. s: control rats, d: 6-OHDA-treated rats, and j: 6-OHDA and desipramine-treated rats. Five rats from each group were used for these experiments. * P,0.05.
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Table 1 MCV, MCH and MCHC levels in 6-OHDA-treated and control rats on day 28 a
6-OHDA rats Control rats a
MCV(fl)
MCH(pg)
MCHC (%)
47.861.8 47.6613.2
18.461.4 19.365.5
38.461.7 40.7612.9
Five rats from each group were used for these experiments.
Fig. 4. Change in WBC levels in 6-OHDA-treated rats. WBC levels were measured in 6-OHDA-treated, 6-OHDA and desipramine-treated, and control rats. s: control rats, d: 6-OHDA-treated rats, and j: 6-OHDA and desipramine-treated rats. Five rats from each group were used for these experiments.
Fig. 6. Circadian rhythm of serum EPO in 6-OHDA-treated and control rats. Circadian rhythm of serum EPO in 6-OHDA-treated rats was monitored on day 7 after 6-OHDA injection and compared with that in control rats. The blood samples were taken from the rats at 1:00, 7:00, 13:00, and 19:00. The bold line indicates the EPO levels in control rats (n55). The circadian rhythm of five different 6-OHDA-treated rats are expressed by the different lines.
3.5. b -adrenergic receptors on erythrocytes treated rats on the same day as the hemogram assay. No significant response in EPO elevation was observed, even after 4 ml of bloodletting in 6-OHDA-treated rats. In contrast, a marked increase in EPO levels was seen after the bloodletting in control rats (Fig. 5). On day 7 after 6-OHDA injection, the circadian rhythm of serum EPO was irregular, compared with that in control rats (Fig. 6).
To study the changes in b-adrenergic receptors on erythrocytes during anemia induced by 6-OHDA administration, specific binding of 125 I-CYP to erythrocytes was calculated as described above. As presented in Fig. 7, 6-OHDA-treated rats showed a significant decrease in b-adrenergic receptors from day 7 to day 28, and reached the normal values after day 35, compared with those in control rats.
4. Discussion
Fig. 5. Changes in EPO levels before and after bloodletting in 6-OHDAtreated rats. The arrow indicates the date of bloodletting. s: control rats, d: 6-OHDA-treated rats. Five rats from each group were used for these experiments. * P,0.05.
We have demonstrated that rats chemically sympathectomized with 6-OHDA showed normocytic and normochromic anemia which was prevented by prior administration of desipramine, a competitive inhibitor of 6-OHDA (Tsao et al., 1996). In addition, since the WBC did not significantly change during 6-OHDA-induced anemia, the effect of 6-OHDA in this experiment may be specific for erythropoiesis, but not for bone marrow suppression. It is well known that 6-OHDA induces various peripheral autonomic dysfunction, such as diarrhea, mydriasis, and a decrease in systolic blood pressure by accumulating the sympathetic nerve terminal and damaging sympathetic nerves via free radical intoxication (Thoenen and Tranzer, 1968). As shown in Fig. 2, significant catecholamine levels were seen, especially on day 7, suggesting adrenergic nerve dysfunction. During the same period of the decrease
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in catecholamine levels, significant hypotension occurred after 6-OHDA treatment, suggesting autonomic dysfunction. Diarrhea was observed from day 3 to around 30 after 6-OHDA injection, which may be further evidence of autonomic dysfunction. It is well known that cardiac output affects renal interstitial PO 2 and has an influence on EPO production (Gaar, 1986). Usually, the cardiac output is enhanced in 6-OHDA-treated animals (Appenroth et al., 1985). However, serum EPO levels did not diminish until day 6, and did not show significant elevation even after bloodletting on day 7 in 6-OHDA-treated rats. These results suggest that renal sympathetic nerve closely connected with EPO regulation. We and other groups previously reported that the anemia associated with neurological disorders, such as FAP, pandysautonomia, and Shy–Drager syndrome (Asahara et al., 1993; Biaggioni et al., 1994; Ando et al., 1996), did not sufficiently increase serum EPO levels, to compensate for anemia which may be a major reason for causing anemia. The results demonstrated in the present experiments were quite consistent with these reports. On the contrary, our preliminary experiments revealed that EPO levels increased due to bloodletting on day 28, when peripheral autonomic functions were recorded (data not shown). These results suggest that EPO secretion may be regulated via renal sympathetic nerves innervated into EPO-secreting cells, and the cells were affected by the procedure of the sympathectomy. In kidney transplant patients, although 10–15% show erythrocytosis for unknown reasons, a significant number of these patients also exhibit low serum EPO levels
independent of renal function (Wickre et al., 1983). In the same way, the circadian rhythm of serum EPO was irregular in 6-OHDA-treated rats. Taken together, the disordered sympathetic nerves may induce the dysregulation of EPO secretion. Since 6-OHDA widely damages the peripheral sympathetic nerves distributed throughout the body, the effect of administered 6-OHDA may not be limited to the sympathetic nerves in the bone marrow and kidneys. Some changes in the metabolism of hormonal and neurotransmitters may occur due to 6-OHDA treatment. Further precise research is needed. As demonstrated in Fig. 7, b-adrenergic receptors on the surface of erythrocytes seemed to be down regulated during 6-OHDA induced anemia. Beckman et al. suggested that the number of b-adrenergic receptors might correlate with that of EPO receptors (Beckman and Fisher, 1979). With decreased numbers of b-adrenergic receptors, EPO receptors may decrease, which may in turn lead to the decreased response of EPO secretion. It seemed important to examine the change in the number of b-adrenergic receptors on the surface of erythroblasts, so we tried to measure them. Unfortunately, because of methodological difficulties, we could not present the established data. It has been well documented that there are a- and b-adrenergic receptors on the surface of erthyrocytes, however, b-receptors are more important in erythropoiesis: Administration of an a-adrenergic stimulant did not elicit the erythropoietic effect, whereas a b-adrenergic stimulant induced erythropoiesis in an in vitro culture of erythroid progenitor cells (Mladenovic and Adamson, 1984). Since
Fig. 7. Changes in b-adrenergic receptors on the surface of erythrocytes during anemia induced by 6-OHDA administration. Five rats from each group were used for these experiments. * P,0.05, ** P,0.01.
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b-adrenergic receptors are abundant on the surface of erthyrocytes, the effect of b-adrenergic blocking agents has often been investigated using erythrocytes (Atlas et al., 1974; Miklavc et al., 1989). In summary, normocytic and normochromic anemia was found in chemically sympathectomized rats treated with 6-OHDA. This anemia was prevented by pre-administration of desipramine, a specific antagonist for 6-OHDA. Serum EPO levels did not respond to the degree of anemia, even after the induction of severe anemia in the 6-OHDA injected rats. Down regulation of b-adrenergic receptors on the surface of erythrocytes occurred in 6-OHDA-treated rats. These results suggest that disordered EPO regulation via disordered sympathetic nerves may induce anemia. 6-OHDA is a useful chemical agent for inducing neurogenic anemia.
Acknowledgements The work was supported by grants from the Amyloidosis Research Committee, the Pathogenesis and Therapy of Hereditary Neuropathy Research Committee, Surveys and Research on Specific Disease of the Ministry of Health and Welfare of Japan.
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Beckman, B., Fisher, J.W., 1979. Changes in beta-2 adrenergic receptor sensitivity with maturation of erythroid progenitor cells. Experientia 35, 1671–1672. Biaggioni, I., Robertson, D., Krants, S., Jones, M., Haile, V., 1994. The anemia of primary autonomic failure and its reversal with recombinant erythropoietin. Ann. Intern. Med. 121, 181–186. Brown, J.E., Adamson, J.W., 1977. The influence of b-adrenergic agonists on erythroid colony formation. J. Clin. Invest. 60, 70–77. Bulloch, K., 1985. Neuroanatomy of lymphoid tissue. In: Guillemin, R. (Ed.), Neural Moduration of Immunity. Raven Press, New York, pp. 111–141. Dresch, C., Minc, J., Poirier, O., Bouvet, D., 1981. Effect of beta adrenergic agonists and blocking agents on hemopoiesis in human bone marrow. Biomedicine 34, 93–98. Erslev, A.J., 1970. Anemia of chronic renal disease. Arch. Intern. Med. 126, 774–780. Eschbach, J.W., Adamson, J.W., 1985. Anemia of end-stage renal disease (ERSD). Kidney Int. 28, 1–5. Gaar, Jr. K.A., 1986. Renal disease and hypertension: the erythrocytosis factor. Med. Hypoth. 19, 359–366. Ganong, W.F., 1973. Biogenic amines, sympathetic nerves and renin secretion. Fed. Proc. 32, 1782–1784. Kostrzewa, R.M., Jacobowitz, D.M., 1974. Pharmacological actions of 6-hydroxydopamine. Pharmacol. Rev. 26, 199–288. McGonigle, R.J., Wallin, J.D., Shadduck, R.K., Fisher, J.W., 1984. Erythropoietin deficiency and inhibition of erythropoiesis in renal insufficiency. Kidney Int. 25, 437–444. Miklavc, A., Kocjan, D., Hadzi, D., Mavri, J., Koller, J., 1989. Binding of agonists and antagonists to beta-adrenergic receptor. Prog. Clin. Biol. Res. 291, 275–280. Mladenovic, J., Adamson, J.W., 1984. Adrenergic modulation of erythropoiesis: in vitro studies of colony-forming cells in normal and polycythaemic man. Br. J. Haematol. 56, 323–332. Prazala, F., Gross, D., Dargon, P., Fisher, J., 1977. Effects of in vitro beta adrenergic activation on rabbit bone marrow erythroid colony-forming cells. Proc. Soci. Exp. Biol. Med. 155, 334–348. Shaw, A.B., 1967. Haemolysis in chronic renal failure. Br. Med. J. 2, 213–216. Takaku, F., Hirashima, K., Okinaka, S., 1961. Effect of bilateral section of the splanchnic nerve on erythropoiesis. Nature 191, 500–501. Thoenen, H., Tranzer, J.P., 1968. Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Naunyn-Schmiedebergs Arch. Pharmacol. 261, 271–288. Tsao, C.W., Cheng, J.T., Shen, C.L., Lin, Y.S., 1996. 6-hydroxydopamine induces thymocyte apoptosis in mice. J. Neuroimmun. 65, 91–95. Wickre, C.G., Norman, D.J., Bennison, A., Barry, J.M., Bennett, W.M., 1983. Postrenal transplant erythrocytosis: a review of 53 patients. Kidney Int. 23, 731–737. Yoshimura, M., Komori, T., Nakanishi, T., Takahashi, H., 1993. Estimation of sulphoconjugated catecholamine concentrations in plasma by high-performance liquid chromatography. Ann. Clin. Biochem. 30, 135–141.
Mechanism of anemia associated with autonomic dysfunction in rats a b, a c c Konen Obayashi , Yukio Ando *, Hisayasu Terazaki , Taro Yamashita , Masaaki Nakamura , Moritaka Suga a , Makoto Uchino c , Masayuki Ando a a
First Department of Internal Medicine, Kumamoto University School of Medicine, 1 -1 -1 Honjo, Kumamoto 860 -0811, Japan b Department of Laboratory Medicine, Kumamoto University School of Medicine, 1 -1 -1 Honjo, Kumamoto 860 -0811, Japan c Department of Neurology, Kumamoto University School of Medicine, 1 -1 -1 Honjo, Kumamoto 860 -0811, Japan Received 20 December 1999; received in revised form 23 February 2000; accepted 23 February 2000
Abstract The aim of this study was to elucidate the mechanism of anemia associated with autonomic dysfunction in rats. Using 6hydroxydopamine (6-OHDA)-treated sympathectomized rats, changes in systolic blood pressure, plasma catecholamine levels, hemograms, erythropoietin (EPO) secretion, and b-adrenergic receptors on erythrocytes were monitored, and compared with desipramine- and 6-OHDA-treated, and control rats. In 6-OHDA-treated rats, systolic blood pressure and plasma catecholamine levels significantly decreased from 7 days after 6-OHDA administration, returning to the control values on day 28. Hemoglobin (Hb), hematocrit (Hct) and red blood cell (RBC) levels significantly decreased from day 14 to day 28, and reached normal values after day 35, but neither corpuscular constants nor white blood cell (WBC) levels changed after anemia occurred. Administration of desipramine 1 day before 6-OHDA injection prevented anemia. EPO levels did not elevate, even after bloodletting to load anemia, and the EPO circadian rhythm was irregular in 6-OHDA-treated rats. b-adrenergic receptors measured using 125 I-cyanopindolol (CYP) significantly decreased from day 7 to day 28, and reached normal values after day 35. These results suggest that irregular EPO secretion via disordered autonomic nerves may induce anemia in patients with autonomic disorders. 2000 Elsevier Science B.V. All rights reserved. Keywords: Anemia; Autonomic dysfunction; 6-OHDA; Erythropoietin; b-adrenergic receptors
1. Introduction Anemia is induced by various factors and pathological conditions, such as lowered red blood cell (RBC) survival time (Erslev, 1970; McGonigle et al., 1984; Eschbach and Adamson, 1985), hemolysis (Shaw, 1967; Eschbach and Adamson, 1985), hemorrhage, iron deficiency, retained toxic substances or uremic inhibitors that interfere with erythroid marrow function (Eschbach and Adamson, 1985) as well as inadequate secretion of erythropoietin (EPO) in the kidneys (Adamson et al., 1968; McGonigle et al.,
Abbreviations: FAP, familial amyloidotic polyneuropathy; ATTR, amyloidogenic transthyretin; Val, valine; Met, methionine; 6-OHDA, 6-hydroxydopamine; Hb, hemoglobin; Hct, hematocrit; RBC, red blood cell; WBC, white blood cell; EPO, erythropoietin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CYP, cyanopindolol *Corresponding author. Tel.: 181-963-735-692; fax: 181-963-735692. E-mail address: [email protected] (Y. Ando)
1984). Owing to the progress of biochemical and molecular genetic methods, the mechanism of several types of anemia has been explained, though many remain to be elucidated. It has been well documented that normocytic and normochromic anemia is sometimes associated with autonomic dysfunction (Asahara et al., 1993; Biaggioni et al., 1994; Ando et al., 1996): we recently reported that anemia was recognized in patients with familial amyloidotic polyneuropathy (FAP) amyloidogenic transthyretin Valine 30 Methionine (ATTR Val 30 Met), pandysautonomia and Shy–Drager syndrome, which are all noted for their severe autonomic dysfunction (Asahara et al., 1993; Ando et al., 1996). Among these three diseases, FAP ATTR Val30 Met shows the most severe autonomic dysfunction, such as orthostatic hypotension, diarrhea, pupillary disorder, decreased sweating, and dry eyes (Ando et al., 1993; Biaggioni et al., 1994), while exhibiting the most severe anemia correlated with the progression of autonomic dysfunction (Asahara et al., 1993; Biaggioni et al., 1994; Ando et al., 1996).
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Since autonomic nerves dominate the kidneys, bone marrow and surrounding tissues, erythropoiesis may be modified by disordered autonomic nerves (Ganong, 1973; Bulloch, 1985). Takaku et al. reported that the reticulocyte response to acute bloodletting was significantly diminished in rats when their kidneys were functionally denerved (Takaku et al., 1961), and there are several reports describing the growth of erythroid progenitor cells being enhanced in the presence of b-adrenergic stimulants in in vitro studies (Brown and Adamson, 1977; Prazala et al., 1977; Beckman and Fisher, 1979; Dresch et al., 1981; Mladenovic and Adamson, 1984). Moreover, Bazan reported the possible relationship between nervous system and hematopoiesis (Bazan, 1991). These results suggest that there is some erythropoietic regulation via the autonomic nervous system. However, the mechanism causing anemia associated with autonomic dysfunction is not well explained. It is also well known that 6-hydroxydopamine (6OHDA) is a useful neurotoxic agent which can reversibly impair the sympathetic nerve terminal (Thoenen and Tranzer, 1968). When it is injected intravenously or intraperitoneally, it accumulates in the peripheral sympathetic nerve terminal and selectively destroys the sympathetic nerves. Its toxic effects directly result from its ability to generate free radical species, and from covalent bonding of quinone oxidation product (Kostrzewa and Jacobowitz, 1974). With this chemical agent, we can create pure sympathectomized rats, which are reversible by the administration of desipramine, a competitive inhibitor of 6-OHDA (Tsao et al., 1996). Moreover, since the neurotoxic effect of 6-OHDA is reversible, we can follow up not only the chemically sympathectomized stage, but also the recovering stage of autonomic dysfunction using 6-OHDAtreated rats. The aim of this study was to elucidate the mechanism of anemia associated with autonomic dysfunction. In 6OHDA-treated rats, the following experiments were performed. (1) To examine the changes in physiological conditions, systolic blood pressure was monitored. (2) To examine the effect of 6-OHDA on catecholamine production in the tissues, plasma epinephrine and norepinephrine levels were measured. (3) To investigate the effect of 6-OHDA on hematopoiesis, changes in hemograms were compared between 6-OHDA-treated and control rats. (4) To investigate the response of EPO to the anemia, changes in serum EPO levels were measured in 6-OHDA-treated rats after bloodletting and compared with those in control rats. (5) To analyze the circadian rhythm of EPO secretion, serum EPO levels were monitored in 6-OHDA-treated rats four times a day and compared with those in control rats. (6) To determine the changes in b-adrenergic receptors on the surface of erythrocytes during anemia, the binding of cyanopindolol (CYP) to erythrocytes was examined in 6-OHDA-treated rats.
2. Materials and methods
2.1. Chemicals 6-OHDA hydrobromide and desipramine were purchased from Sigma Chemical (St. Louis, USA). 125 I-CYP reagent (2,200 Ci / mmol) was obtained from New England Nuclear (Boston, MA, USA). All chemicals used in this study were of analytical grade.
2.2. In vivo experiments To omit the androgenic effect on the secretion of EPO, 30 female Wistar rats (7-week-old, 150–200 g) were used in this study. These rats were fed a laboratory pellet diet and water, and were maintained under the same conditions. 6-OHDA was dissolved in saline containing 0.1% ascorbic acid immediately prior to injection. Fifteen rats were chemically sympathectomized via intravenous administration of 0.1 ml of 6-OHDA solution (containing 100 mg / kg of 6-OHDA). Another ten rats were administrated an equivalent volume of a vehicle (0.1 ml, iv) following the same procedure. Five of the 6-OHDA-treated rats were simultaneously injected with 0.1 ml of desipramine in saline (10 mg / kg) 1 day before 6-OHDA injection (Thoenen and Tranzer, 1968).
2.3. Systolic blood pressure Systolic blood pressure was monitored in fifteen 6OHDA injected rats at the tail using a blood pressure monitor, model PS-200S (Riken, Tokyo, Japan) from day 0 to 42.
2.4. Measurement of plasma epinephrine and norepinephrine levels in the 6 -OHDA-treated rats’ serum Three hundred microliters of plasma samples from 6OHDA-treated, 6-OHDA and desipramine-treated rats, and control rats were mixed with 300 ml of 6% perchloric acid solution in a vortex-mixer for deproteination. After adding 200 ml of 1.5 mol / l sodium acetate, the mixture was stirred and centrifuged (3000 rpm) at 28C for 20 min. The clear supernatant was applied to the autosampler of a high performance liquid chromatography (HPLC) analyzer (Tosoh, Tokyo, Japan; Eurogenetics, Tessenderlo, Belgium; Tosoh Medics, Emeryville, CA, USA) (Yoshimura et al., 1993).
2.5. Hemograms After collecting 0.2 ml of blood samples, hemoglobin (Hb), hematocrit (Hct), red blood cell (RBC) and white blood cell (WBC) levels were measured in five 6-OHDAtreated rats, five desipramine and 6-OHDA-treated rats,
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and five control rats on day 0, 3, 7, 14, 21, 28, 35 and 42 after treatment. In addition, we calculated the corpuscular constants [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)] from this data on day 28.
2.6. Serum EPO assay Serum EPO levels were measured in five 6-OHDAtreated rats and five control rats after 4 ml of bloodletting on day 7. The circadian rhythm of EPO secretion was determined in five 6-OHDA-treated rats and compared with that in five control rats. The blood samples were taken from the rats at 1: 00, 7: 00, 13: 00, and 19: 00. The EPO levels were measured using an EPO-specific ELISA kit (Kyokuto, Osaka, Japan).
2.7. b -adrenergic receptors on erythrocytes The number of b-adrenergic receptors was determined in the blood samples in five 6-OHDA injected rats and five control rats on day 0, 3, 7, 14, 21, 28, 35 and 42 after treatment. Binding of 125 I-CYP to erythrocytes was determined by incubating 5310 5 cells with 0.02 mCi of 125 I-CYP in 150 mM sodium chloride, 1 mM ascorbic acid, and 20 mM Tris, pH 7.4, for 90 min at 308C. Receptor density was determined by incubating the erythrocytes with at least five different concentrations of 125 ICYP, ranging from 25 to 200 pM. Non-specific binding was determined by measuring the amount of 125 I-CYP bound to the cells in the presence of 6 mM DL-propranolol. The experiments were terminated by dilution with 5 ml of ice-cold incubation buffer followed by vacuum filtration through Whatman GF / C filters. The sample tubes were rinsed with an additional 5 ml of the cold buffer and the filters were washed with an additional 30 ml of the icecold incubation buffer. The filters were packed into sample tubes and counted in a gamma counter, model ARC-361 (Aloka, Tokyo, Japan). The count per min (cpm) reflecting the real receptor density was corrected by deleting the non-specific binding values as described above.
2.8. Statistical analysis All the data were expressed as mean6S.D. The significance of the data was determined by unpaired Student’s t-test. P values less than 0.05 were considered significant.
3. Results
3.1. Change in systolic blood pressure after 6 -OHDA treatment To investigate changes in physiological conditions after
Fig. 1. Changes in systolic blood pressure in 6-OHDA-treated rats. Systolic blood pressure was monitored in the tail as described previously. The statistical significance was compared before and after administration of 6-OHDA. Fifteen rats were used for these experiments. * P,0.05.
6-OHDA injection, systolic blood pressure was monitored as demonstrated in Fig. 1. A significant decrease in blood pressure was observed from day 4 to 21 after 6-OHDA administration (P,0.05), and reached a normal value after day 28.
3.2. Measurement of plasma epinephrine and norepinephrine levels in the 6 -OHDA-treated rats’ serum To examine the effect of 6-OHDA on catecholamine production in the tissues, plasma epinephrine and norepinephrine levels were measured. As demonstrated in Fig. 2, both plasma epinephrine and norepinephrine levels significantly decreased in 6-OHDA-treated rats on day 7 after 6-OHDA treatment, returning to the control values on day 28. Desipramine injection effectively prevented the decrease in catecholamine levels.
3.3. Changes in hemograms To elucidate the effect of 6-OHDA on hematopoiesis, Hb, Hct, RBC, and WBC levels were measured in 6OHDA-treated rats and compared with those in control rats. Hb, Hct and RBC levels significantly decreased from day 14 to 28 (P,0.05), and reached the normal values after day 35 in 6-OHDA-treated rats. Desipramine intravenously administered 1 day before the 6-OHDA in-
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Fig. 2. Measurement of plasma epinephrine (A) and norepinephrine (B) levels in 6-OHDA-treated rats’ serum. Plasma epinephrine and norepinephrine levels were measured in 6-OHDA-treated, 6-OHDA- and desipramine-treated, and control rats. h: control rats, j: 6-OHDA-treated rats, and : 6-OHDA and desipramine-treated rats. Five rats from each group were used for these experiments. * P,0.05.
jection effectively prevented the occurrence of anemia (Fig. 3 A-C). Neither MCV, MCH, MCHC nor WBC levels prominently changed, even after anemia occurred in 6OHDA injected rats (Table 1, Fig. 4).
3.4. Changes in EPO secretion during anemia To check the EPO response in 6-OHDA-induced anemia, serum EPO levels were measured in 6-OHDA-
Fig. 3. Change in the hemograms in 6-OHDA-treated rats. Hb (A), Hct (B), and RBC (C) levels were measured in 6-OHDA-treated, 6-OHDA and desipramine-treated, and control rats. s: control rats, d: 6-OHDA-treated rats, and j: 6-OHDA and desipramine-treated rats. Five rats from each group were used for these experiments. * P,0.05.
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Table 1 MCV, MCH and MCHC levels in 6-OHDA-treated and control rats on day 28 a
6-OHDA rats Control rats a
MCV(fl)
MCH(pg)
MCHC (%)
47.861.8 47.6613.2
18.461.4 19.365.5
38.461.7 40.7612.9
Five rats from each group were used for these experiments.
Fig. 4. Change in WBC levels in 6-OHDA-treated rats. WBC levels were measured in 6-OHDA-treated, 6-OHDA and desipramine-treated, and control rats. s: control rats, d: 6-OHDA-treated rats, and j: 6-OHDA and desipramine-treated rats. Five rats from each group were used for these experiments.
Fig. 6. Circadian rhythm of serum EPO in 6-OHDA-treated and control rats. Circadian rhythm of serum EPO in 6-OHDA-treated rats was monitored on day 7 after 6-OHDA injection and compared with that in control rats. The blood samples were taken from the rats at 1:00, 7:00, 13:00, and 19:00. The bold line indicates the EPO levels in control rats (n55). The circadian rhythm of five different 6-OHDA-treated rats are expressed by the different lines.
3.5. b -adrenergic receptors on erythrocytes treated rats on the same day as the hemogram assay. No significant response in EPO elevation was observed, even after 4 ml of bloodletting in 6-OHDA-treated rats. In contrast, a marked increase in EPO levels was seen after the bloodletting in control rats (Fig. 5). On day 7 after 6-OHDA injection, the circadian rhythm of serum EPO was irregular, compared with that in control rats (Fig. 6).
To study the changes in b-adrenergic receptors on erythrocytes during anemia induced by 6-OHDA administration, specific binding of 125 I-CYP to erythrocytes was calculated as described above. As presented in Fig. 7, 6-OHDA-treated rats showed a significant decrease in b-adrenergic receptors from day 7 to day 28, and reached the normal values after day 35, compared with those in control rats.
4. Discussion
Fig. 5. Changes in EPO levels before and after bloodletting in 6-OHDAtreated rats. The arrow indicates the date of bloodletting. s: control rats, d: 6-OHDA-treated rats. Five rats from each group were used for these experiments. * P,0.05.
We have demonstrated that rats chemically sympathectomized with 6-OHDA showed normocytic and normochromic anemia which was prevented by prior administration of desipramine, a competitive inhibitor of 6-OHDA (Tsao et al., 1996). In addition, since the WBC did not significantly change during 6-OHDA-induced anemia, the effect of 6-OHDA in this experiment may be specific for erythropoiesis, but not for bone marrow suppression. It is well known that 6-OHDA induces various peripheral autonomic dysfunction, such as diarrhea, mydriasis, and a decrease in systolic blood pressure by accumulating the sympathetic nerve terminal and damaging sympathetic nerves via free radical intoxication (Thoenen and Tranzer, 1968). As shown in Fig. 2, significant catecholamine levels were seen, especially on day 7, suggesting adrenergic nerve dysfunction. During the same period of the decrease
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in catecholamine levels, significant hypotension occurred after 6-OHDA treatment, suggesting autonomic dysfunction. Diarrhea was observed from day 3 to around 30 after 6-OHDA injection, which may be further evidence of autonomic dysfunction. It is well known that cardiac output affects renal interstitial PO 2 and has an influence on EPO production (Gaar, 1986). Usually, the cardiac output is enhanced in 6-OHDA-treated animals (Appenroth et al., 1985). However, serum EPO levels did not diminish until day 6, and did not show significant elevation even after bloodletting on day 7 in 6-OHDA-treated rats. These results suggest that renal sympathetic nerve closely connected with EPO regulation. We and other groups previously reported that the anemia associated with neurological disorders, such as FAP, pandysautonomia, and Shy–Drager syndrome (Asahara et al., 1993; Biaggioni et al., 1994; Ando et al., 1996), did not sufficiently increase serum EPO levels, to compensate for anemia which may be a major reason for causing anemia. The results demonstrated in the present experiments were quite consistent with these reports. On the contrary, our preliminary experiments revealed that EPO levels increased due to bloodletting on day 28, when peripheral autonomic functions were recorded (data not shown). These results suggest that EPO secretion may be regulated via renal sympathetic nerves innervated into EPO-secreting cells, and the cells were affected by the procedure of the sympathectomy. In kidney transplant patients, although 10–15% show erythrocytosis for unknown reasons, a significant number of these patients also exhibit low serum EPO levels
independent of renal function (Wickre et al., 1983). In the same way, the circadian rhythm of serum EPO was irregular in 6-OHDA-treated rats. Taken together, the disordered sympathetic nerves may induce the dysregulation of EPO secretion. Since 6-OHDA widely damages the peripheral sympathetic nerves distributed throughout the body, the effect of administered 6-OHDA may not be limited to the sympathetic nerves in the bone marrow and kidneys. Some changes in the metabolism of hormonal and neurotransmitters may occur due to 6-OHDA treatment. Further precise research is needed. As demonstrated in Fig. 7, b-adrenergic receptors on the surface of erythrocytes seemed to be down regulated during 6-OHDA induced anemia. Beckman et al. suggested that the number of b-adrenergic receptors might correlate with that of EPO receptors (Beckman and Fisher, 1979). With decreased numbers of b-adrenergic receptors, EPO receptors may decrease, which may in turn lead to the decreased response of EPO secretion. It seemed important to examine the change in the number of b-adrenergic receptors on the surface of erythroblasts, so we tried to measure them. Unfortunately, because of methodological difficulties, we could not present the established data. It has been well documented that there are a- and b-adrenergic receptors on the surface of erthyrocytes, however, b-receptors are more important in erythropoiesis: Administration of an a-adrenergic stimulant did not elicit the erythropoietic effect, whereas a b-adrenergic stimulant induced erythropoiesis in an in vitro culture of erythroid progenitor cells (Mladenovic and Adamson, 1984). Since
Fig. 7. Changes in b-adrenergic receptors on the surface of erythrocytes during anemia induced by 6-OHDA administration. Five rats from each group were used for these experiments. * P,0.05, ** P,0.01.
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b-adrenergic receptors are abundant on the surface of erthyrocytes, the effect of b-adrenergic blocking agents has often been investigated using erythrocytes (Atlas et al., 1974; Miklavc et al., 1989). In summary, normocytic and normochromic anemia was found in chemically sympathectomized rats treated with 6-OHDA. This anemia was prevented by pre-administration of desipramine, a specific antagonist for 6-OHDA. Serum EPO levels did not respond to the degree of anemia, even after the induction of severe anemia in the 6-OHDA injected rats. Down regulation of b-adrenergic receptors on the surface of erythrocytes occurred in 6-OHDA-treated rats. These results suggest that disordered EPO regulation via disordered sympathetic nerves may induce anemia. 6-OHDA is a useful chemical agent for inducing neurogenic anemia.
Acknowledgements The work was supported by grants from the Amyloidosis Research Committee, the Pathogenesis and Therapy of Hereditary Neuropathy Research Committee, Surveys and Research on Specific Disease of the Ministry of Health and Welfare of Japan.
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