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Effects of space flight stress on proopiomelanocortin, proenkephalin A, and tachykinin neuropeptidergic systems in the rat posterior pituitary
Life Sciences, Vol. 55, No. 5, pp. 347-350, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00
Pergamon 0024-3205(94)00127.8
EFFECTS OF SPACE F L I G H T STRESS ON PROOPIOMELANOCORTIN, PROENKEPHALIN A, AND TACHYKININ NEUROPEPTIDERGIC SYSTEMS IN THE RAT POSTERIOR PITUITARY 1Xuegong Zhu and 1,2,3,*Dominic M. Desiderio 1Charles B. Stout Neuroscience Mass Spectrometry Laboratory, Departments of 2Biochemistry and 3Neurology, The University of Tennessee, Memphis, 800 Madison Avenue, Memphis, Tennessee 38163 (USA) (Received in final form May 20, 1994)
Summary 13-endorphin-like immunoreactivity (BE-li), methionine enkephalin-like immunoreactivity (ME-Ii), and substance P-like immunoreactivity (SP-li) were measured in the posterior pituitary of rats that experienced a 5-day space flight in a Space Shuttle. ME-li and SP-li were both significantly lower compared to the control rats. However, there was no difference in BE-Ii between flight and control rats. These data suggest that the space flight stress diminished the methionine enkephalin (ME) and substance P (SP) concentrations in the posterior pituitary without affecting the ~-endorphin (BE) concentration. Thus, the proenkephalin A and tachykinin, but not proopiomelanocortin, neuropeptidergic systems in the posterior pituitary may respond to this type of unique stress. Key Words: space flight stress, proopiomelanocortin,proenkephalin A, tachykinin peptide, pituitary
It is well-known that stress, including infectious diseases, psychiatric disorders, surgical trauma, and strenuous exercises, increases the secretion of the proopiomelanocortin peptide BE and the proenkephalin A peptide ME into the blood circulation (5). The secreted opioid peptides may then affect various behavior responses, such as pain perception, motility, fighting, and physiological responses (8). The tachykinin peptide SP is also considered to be an important neuropeptide in the stress process, and has been demonstrated to normalize stress-induced disorders (7). In rats, BE has been localized in the corticotrophs of the anterior pituitary and melanotrophs of the neurointermediate pituitary, but little in the posterior pituitary (1). ME has been localized in the somatotrophs of the anterior pituitary (9) and in the posterior pituitary (2). SP has been localized in the SP-Ii fibers and gland cells in the anterior lobe and in the SP-li fibers in the posterior pituitary (4). In this communication, we report the radioimmunoassay measurements of BE, ME, and SP in the posterior pituitary from rats who had experienced a 5-day space flight in a Space Shuttle by *Corresponding author Dr. Dominic Desiderio, The University of Tennessee, Memphis, Charles B. Stout Neuroscience Mass Spectrometry Laboratory, 956 Court Avenue, Room A-218, Memphis, TN 38163. Telephone: (901) 448-5488; Telefax: (901) 448-7842; Bitnet: DDESIDERIO@UTMEM1
348
Effects of Space Flight Stress
Vol. 55, No. 5, 1994
the National Aeronautics and Space Administration (NASA). Because this research is part of a tissue-sharing program by NASA, the posterior pituitary is the only tissue available to this laboratory. During the space flight, these rats had been subjected to three stages of stress: the enormous acceleration at the launching of the Space Shuttle, living in a microgravity force for 5 days, and the enormous deceleration during the landing process of the Space Shuttle (deceleration in space, rapid descent, near-earth approach, and actual stopping on earth). A comparison was made between the measurements of BE-li, ME-li, and SP-Ii in the posterior pituitary of the Space Shuttle rats to those of control rats that remained on Earth. Materials and methods
Rat posterior pituitary. Male Sprague-Dawley rats (Taconic Farms, Inc., Germantown, NY) were housed in standard vivarium cages at the Kennedy Space Center, with access to ARC foodbar diet and water ad libitum. The animals were preconditioned to about 28 °C, which mimicked the temperature in the animal housing in the Space Shuttle. The rats weighed about 200 g each at launch. A single housing with 6 rats was flown. After a 5-day space flight (5 d, 9 h, 23 min, 32 see), the posterior pituitary was dissected from the rats 5-12 h after landing under anesthesia (acepromazine, 10 mg/kg), frozen immediately in liquid nitrogen, and shipped to our laboratory on dry-ice via an air shipping service. The control rats were from the same litter and in the same housing at the Kennedy Space Center. The control rats were dissected 24-30 hour after the Space Shuttle landed. After centrifugation (31,000 x g) of the homogenate for 30 min at 4 °C, the supernatant was collected. Tissue processing. The posterior pituitary tissues from 5 flight and 5 control rats were homogenized individually in 10 ml acetic acid (1N; w:v = 1:100), and an aliquot (5%) was taken for total protein determination by a modified Lowry micro-colorimetric total protein kit 690A from Sigma Chemical Company (St. Louis, MO). Rather than adding any enzyme inhibitors before tissue homogenization, acidification plus a rapid lowering of the temperature stopped enzymatic processing (10). Sep-Pak. An octadecylsilyl (ODS) mini-column (Sep-Pak ® cartridge, Millipore-Waters, Milford, M.A) was used to remove water-soluble solutes, proteins, saccharides, etc., from the tissue homogenate (3). The entire supernatant from each tissue was applied to an ODS mini-column, which was previously washed with 4 ml of 0.1% trifluoroacetic acid (TFA). The peptide-rich fraction was removed with a bolus of acetonitrile (3 ml, 100%). The acetonitrile was evaporated, and the sample was lyophilized to dryness. Radioimmunoassay (RIA). Commercial RIA kits (IncStar, Stillwater, MN) were used to measure ME-li, SP-li, and BE-Ii. The cross-reactivities of the antibody in the ME kit towards synthetic peptides were 2.8% for leucine enkephalin (LE), 0.10% for ct-endorphin (B-lipotropin61_ 77 (13-LPH61-77)), and <0.002% for SP, BE, porcine dynorphinl_13, and ct-neo-endorphin. The cross-reactivities of the antibody in the SP kit toward synthetic peptides were <0.002% for ME, LE, eledoisin, and physalemin; and 0.008% for BE. The cross-reactivities of the antibody in the BE kit toward synthetic peptides were 100% for human and rat BE, 5.6% for human 13-LPH, and <0.1% for dynorphin, a-neo-endorphin, a-endorphin, LE, ME, a-endorphin (I]-LPH61-76), ctendorphin (13-LPH61-77),adrenocorticotropic hormonel.39 (ACTH1-39), ACTHI-24, ct-melanocytestimulating hormone (ct-MSH), 13-MSH, prolactin, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, vasopressin, and oxytocin. Statistical analysis. A two-sample t-test procedure using SAS software (SAS Institute, Cary, NC) was performed on these data to test for statistical significance. A significance level of P = 0.05 was used for comparison. Results Figure 1 plots the RIA results of BE-li, ME-Ii, and SP-li in the posterior pituitary from the control and Space Shuttle rats. ME-Ii and SP-Ii were both significantly lower than the control (P < 0.05). However, there was no difference in the BE-li measurements (P > 0.05).
Vol. 55, No. 5, 1994
Effects of Space Flight Stress
1500
4000
349
6.00
3000 1000 =
T
4.00
T
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"& X-X-X` x-x`x x. x. x-.xx,
2000 x x x x x x
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Fig. 1 Measurements of ME-Ii, SP-li, and BE-Ii in the posterior pituitary of the control and Space Shuttle rats. Data are given as the average neuropeptide-li/mg protein + standard error of the mean for five pituitary tissues.
The range of the neuropeptide-like immunoreactivity measurements are: BE-Ii (control) 2.75-4.33 pmol/mg protein, BE-Ii (flight) 3.29-4.51 pmol/mg protein; ME-li (control) 467-5227 pmol/mg protein, ME-li (flight) 34.5-109 pmol/mg protein; SP-li (control) 704-1280 fmol/mg protein, SP-li (flight) 288-523 fmol/mg protein. Discussion Our data show that the amount of ME-li and SP-li were diminished in the posterior pituitary in the Space Shuttle rats, implicating that the space flight stress mobilized ME and SP in the posterior pituitary, but did not affect BE in the posterior pituitary. Although stress usually enhances the secretion o f BE from the anterior and neurointermediate lobes, our data suggest that stress would not affect the BE concentration in the posterior pituitary. SP has been shown to be an important ann-stressor, able to normalize the functional disorders accompanying chronic stress (7). In fact, SP-li was found to be diminished in rat adrenals and pituitary after chronic stress (6). The mobilization of the proenkephalin A and tachykininergic systems in the Space Shuttle rat posterior pituitaries could have involved any one or several of the metabolic steps: D N A to R N A to preprohormone precursor molecule to various intermediate-sized precursor molecules to neuropeptide to inactive metabolites. An increased level of a neuropeptide could be the result of an increase in its synthesis and/or a decrease in its inactivation. Further, stress could effect the production of the enzymes that mediate each one of these metabolism points.
350
Effects of Space Flight Stress
Vol. 55, No. 5, 1994
Acknowledements The authors gratefully acknowledge financial support from the National Institutes of Health (NIH GM 26666 to DMD) and the University of Tennessee Center in Excellence for Neuroscienee (predoctoral fellowship to XZ). We thank the National Aeronautics and Space Administration (NASA) for providing us with the animal tissues for this research. References 1. F. BLOOM, E. BATYENBERG, J. ROSSIER, N. LING, J. LEPPALUOTO, T. M. VARGO and R. GUILLEMIN, Life Sci. 20 43-48 (1977). 2. T. DUKA, V. HOLLT, R. PRZEWLOCKI and D. WESCHE, Biochem. Biophys. Res. Commun. 85 1119-1127 (1978). 3. T. HIGA and D. M. DESIDERIO, Int. J. Peptide Protein Res. 33 250-255 (1989). 4. J. D. MIKKELSEN, P. J. LARSEN, M. MOLLER, H. VILHARDT and T. S(ERMARK, Neuroendocrinol. 50 100-108 (1989). 5. M.J. MILLAN and A. HERZ, Int. Rev. Neurobiol. 26 1-83 (1985). 6. K. NIEBER, I. ROSKE, R. RATHSACK and P. OEHME, Endocrinol. Exp. 24 47-54 (1990). 7. P. OEHME, K. HECHT, H. D. FAULHABER, K. NIEBER, I. ROSKE and R. RATHSACK, J. Cardiov. Pharmacol. 10 S 109-S 111 (1987). 8. G . A . OLSON, R. D. OLSON and A. J. KASTIN, Peptides 8 1135-1164 (1987). 9. E. WEBER, K. H. VOIGT and R. MARTIN, Proc. Natl. Acad. Sci. USA 7._556134-6138 (1978). 10. X. ZHU and D. M. DESIDERIO, Anal. Lett. Submitted.
Pergamon 0024-3205(94)00127.8
EFFECTS OF SPACE F L I G H T STRESS ON PROOPIOMELANOCORTIN, PROENKEPHALIN A, AND TACHYKININ NEUROPEPTIDERGIC SYSTEMS IN THE RAT POSTERIOR PITUITARY 1Xuegong Zhu and 1,2,3,*Dominic M. Desiderio 1Charles B. Stout Neuroscience Mass Spectrometry Laboratory, Departments of 2Biochemistry and 3Neurology, The University of Tennessee, Memphis, 800 Madison Avenue, Memphis, Tennessee 38163 (USA) (Received in final form May 20, 1994)
Summary 13-endorphin-like immunoreactivity (BE-li), methionine enkephalin-like immunoreactivity (ME-Ii), and substance P-like immunoreactivity (SP-li) were measured in the posterior pituitary of rats that experienced a 5-day space flight in a Space Shuttle. ME-li and SP-li were both significantly lower compared to the control rats. However, there was no difference in BE-Ii between flight and control rats. These data suggest that the space flight stress diminished the methionine enkephalin (ME) and substance P (SP) concentrations in the posterior pituitary without affecting the ~-endorphin (BE) concentration. Thus, the proenkephalin A and tachykinin, but not proopiomelanocortin, neuropeptidergic systems in the posterior pituitary may respond to this type of unique stress. Key Words: space flight stress, proopiomelanocortin,proenkephalin A, tachykinin peptide, pituitary
It is well-known that stress, including infectious diseases, psychiatric disorders, surgical trauma, and strenuous exercises, increases the secretion of the proopiomelanocortin peptide BE and the proenkephalin A peptide ME into the blood circulation (5). The secreted opioid peptides may then affect various behavior responses, such as pain perception, motility, fighting, and physiological responses (8). The tachykinin peptide SP is also considered to be an important neuropeptide in the stress process, and has been demonstrated to normalize stress-induced disorders (7). In rats, BE has been localized in the corticotrophs of the anterior pituitary and melanotrophs of the neurointermediate pituitary, but little in the posterior pituitary (1). ME has been localized in the somatotrophs of the anterior pituitary (9) and in the posterior pituitary (2). SP has been localized in the SP-Ii fibers and gland cells in the anterior lobe and in the SP-li fibers in the posterior pituitary (4). In this communication, we report the radioimmunoassay measurements of BE, ME, and SP in the posterior pituitary from rats who had experienced a 5-day space flight in a Space Shuttle by *Corresponding author Dr. Dominic Desiderio, The University of Tennessee, Memphis, Charles B. Stout Neuroscience Mass Spectrometry Laboratory, 956 Court Avenue, Room A-218, Memphis, TN 38163. Telephone: (901) 448-5488; Telefax: (901) 448-7842; Bitnet: DDESIDERIO@UTMEM1
348
Effects of Space Flight Stress
Vol. 55, No. 5, 1994
the National Aeronautics and Space Administration (NASA). Because this research is part of a tissue-sharing program by NASA, the posterior pituitary is the only tissue available to this laboratory. During the space flight, these rats had been subjected to three stages of stress: the enormous acceleration at the launching of the Space Shuttle, living in a microgravity force for 5 days, and the enormous deceleration during the landing process of the Space Shuttle (deceleration in space, rapid descent, near-earth approach, and actual stopping on earth). A comparison was made between the measurements of BE-li, ME-li, and SP-Ii in the posterior pituitary of the Space Shuttle rats to those of control rats that remained on Earth. Materials and methods
Rat posterior pituitary. Male Sprague-Dawley rats (Taconic Farms, Inc., Germantown, NY) were housed in standard vivarium cages at the Kennedy Space Center, with access to ARC foodbar diet and water ad libitum. The animals were preconditioned to about 28 °C, which mimicked the temperature in the animal housing in the Space Shuttle. The rats weighed about 200 g each at launch. A single housing with 6 rats was flown. After a 5-day space flight (5 d, 9 h, 23 min, 32 see), the posterior pituitary was dissected from the rats 5-12 h after landing under anesthesia (acepromazine, 10 mg/kg), frozen immediately in liquid nitrogen, and shipped to our laboratory on dry-ice via an air shipping service. The control rats were from the same litter and in the same housing at the Kennedy Space Center. The control rats were dissected 24-30 hour after the Space Shuttle landed. After centrifugation (31,000 x g) of the homogenate for 30 min at 4 °C, the supernatant was collected. Tissue processing. The posterior pituitary tissues from 5 flight and 5 control rats were homogenized individually in 10 ml acetic acid (1N; w:v = 1:100), and an aliquot (5%) was taken for total protein determination by a modified Lowry micro-colorimetric total protein kit 690A from Sigma Chemical Company (St. Louis, MO). Rather than adding any enzyme inhibitors before tissue homogenization, acidification plus a rapid lowering of the temperature stopped enzymatic processing (10). Sep-Pak. An octadecylsilyl (ODS) mini-column (Sep-Pak ® cartridge, Millipore-Waters, Milford, M.A) was used to remove water-soluble solutes, proteins, saccharides, etc., from the tissue homogenate (3). The entire supernatant from each tissue was applied to an ODS mini-column, which was previously washed with 4 ml of 0.1% trifluoroacetic acid (TFA). The peptide-rich fraction was removed with a bolus of acetonitrile (3 ml, 100%). The acetonitrile was evaporated, and the sample was lyophilized to dryness. Radioimmunoassay (RIA). Commercial RIA kits (IncStar, Stillwater, MN) were used to measure ME-li, SP-li, and BE-Ii. The cross-reactivities of the antibody in the ME kit towards synthetic peptides were 2.8% for leucine enkephalin (LE), 0.10% for ct-endorphin (B-lipotropin61_ 77 (13-LPH61-77)), and <0.002% for SP, BE, porcine dynorphinl_13, and ct-neo-endorphin. The cross-reactivities of the antibody in the SP kit toward synthetic peptides were <0.002% for ME, LE, eledoisin, and physalemin; and 0.008% for BE. The cross-reactivities of the antibody in the BE kit toward synthetic peptides were 100% for human and rat BE, 5.6% for human 13-LPH, and <0.1% for dynorphin, a-neo-endorphin, a-endorphin, LE, ME, a-endorphin (I]-LPH61-76), ctendorphin (13-LPH61-77),adrenocorticotropic hormonel.39 (ACTH1-39), ACTHI-24, ct-melanocytestimulating hormone (ct-MSH), 13-MSH, prolactin, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, vasopressin, and oxytocin. Statistical analysis. A two-sample t-test procedure using SAS software (SAS Institute, Cary, NC) was performed on these data to test for statistical significance. A significance level of P = 0.05 was used for comparison. Results Figure 1 plots the RIA results of BE-li, ME-Ii, and SP-li in the posterior pituitary from the control and Space Shuttle rats. ME-Ii and SP-Ii were both significantly lower than the control (P < 0.05). However, there was no difference in the BE-li measurements (P > 0.05).
Vol. 55, No. 5, 1994
Effects of Space Flight Stress
1500
4000
349
6.00
3000 1000 =
T
4.00
T
k'gx7
"& X-X-X` x-x`x x. x. x-.xx,
2000 x x x x x x
500 -
xx
~.
x--%\
X` X - X ` x. x. x` N. x . N .
m
2.00 x~x _._ ~ \\XX`\X` X`X`X-
X - X-'N. X-\X-
x ~ x !
X-\X XN~ x x x
x` x` xx` x`x\ \ \ X`\X` X-\\
1000
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NX`X` !
!
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X-X`\ ~XX XXX ~X`XX`X`XXXX X~X xxx XXX x x x XXX \ \ \ ~X`X X`X`XX`X`X-
!
I
e
~
tm
e
,-v
Fig. 1 Measurements of ME-Ii, SP-li, and BE-Ii in the posterior pituitary of the control and Space Shuttle rats. Data are given as the average neuropeptide-li/mg protein + standard error of the mean for five pituitary tissues.
The range of the neuropeptide-like immunoreactivity measurements are: BE-Ii (control) 2.75-4.33 pmol/mg protein, BE-Ii (flight) 3.29-4.51 pmol/mg protein; ME-li (control) 467-5227 pmol/mg protein, ME-li (flight) 34.5-109 pmol/mg protein; SP-li (control) 704-1280 fmol/mg protein, SP-li (flight) 288-523 fmol/mg protein. Discussion Our data show that the amount of ME-li and SP-li were diminished in the posterior pituitary in the Space Shuttle rats, implicating that the space flight stress mobilized ME and SP in the posterior pituitary, but did not affect BE in the posterior pituitary. Although stress usually enhances the secretion o f BE from the anterior and neurointermediate lobes, our data suggest that stress would not affect the BE concentration in the posterior pituitary. SP has been shown to be an important ann-stressor, able to normalize the functional disorders accompanying chronic stress (7). In fact, SP-li was found to be diminished in rat adrenals and pituitary after chronic stress (6). The mobilization of the proenkephalin A and tachykininergic systems in the Space Shuttle rat posterior pituitaries could have involved any one or several of the metabolic steps: D N A to R N A to preprohormone precursor molecule to various intermediate-sized precursor molecules to neuropeptide to inactive metabolites. An increased level of a neuropeptide could be the result of an increase in its synthesis and/or a decrease in its inactivation. Further, stress could effect the production of the enzymes that mediate each one of these metabolism points.
350
Effects of Space Flight Stress
Vol. 55, No. 5, 1994
Acknowledements The authors gratefully acknowledge financial support from the National Institutes of Health (NIH GM 26666 to DMD) and the University of Tennessee Center in Excellence for Neuroscienee (predoctoral fellowship to XZ). We thank the National Aeronautics and Space Administration (NASA) for providing us with the animal tissues for this research. References 1. F. BLOOM, E. BATYENBERG, J. ROSSIER, N. LING, J. LEPPALUOTO, T. M. VARGO and R. GUILLEMIN, Life Sci. 20 43-48 (1977). 2. T. DUKA, V. HOLLT, R. PRZEWLOCKI and D. WESCHE, Biochem. Biophys. Res. Commun. 85 1119-1127 (1978). 3. T. HIGA and D. M. DESIDERIO, Int. J. Peptide Protein Res. 33 250-255 (1989). 4. J. D. MIKKELSEN, P. J. LARSEN, M. MOLLER, H. VILHARDT and T. S(ERMARK, Neuroendocrinol. 50 100-108 (1989). 5. M.J. MILLAN and A. HERZ, Int. Rev. Neurobiol. 26 1-83 (1985). 6. K. NIEBER, I. ROSKE, R. RATHSACK and P. OEHME, Endocrinol. Exp. 24 47-54 (1990). 7. P. OEHME, K. HECHT, H. D. FAULHABER, K. NIEBER, I. ROSKE and R. RATHSACK, J. Cardiov. Pharmacol. 10 S 109-S 111 (1987). 8. G . A . OLSON, R. D. OLSON and A. J. KASTIN, Peptides 8 1135-1164 (1987). 9. E. WEBER, K. H. VOIGT and R. MARTIN, Proc. Natl. Acad. Sci. USA 7._556134-6138 (1978). 10. X. ZHU and D. M. DESIDERIO, Anal. Lett. Submitted.