Methantmts o/Ageing anti De~elopmettt 60 ( 1991 ) I ]5-142 Elsevier Soentlfic Pubhshers Ireland Ltd

1"~5

CHRONIC HYPOXIA INCREASES ~-ADRENERGIC RECEPTOR DENSITY IN THE LUNGS OF YOUNG AND OLD RATS

DAVID J BIRNKRANT a, SCOTT L MADER b, ERIK VAN LUNTEREN b and PAMELA B DAVIS db Departments of " Pedtatrtt s and hMedtt me Case Western Reserve Umverwt) S( hool o/Metht tnc Rambo)t Babtes and Chddrena Hospttal, Cleveland OH ( U S A)

(Received December 31st, 1990)

SUMMARY

To test the hypothesis that the abdlty to regulate/3-adrenerglc receptor (BAR) density m response to chromc hypoxlc stress ~s ~mpalred by agmg, we measured BAR density m the lungs of young (age 3 months) and aged (age 20 months) rats exposed to hypobanc hypoxm (1/2 atm) for 3 weeks BAR density increased by 63% m the lungs of both young and aged rats exposed to chromc hypoxm Lung BAR density was unaffected by aging, independent of hypoxlc condmons We conclude that the abd~ty to respond to chronic hypoxlc stress with increased lung BAR density is unaffected by aging m rats

Key words /3-Adrenerglc responses, Hypoxla, Aging, Rat lung

INTRODUCTION

The decreased abdlty to adapt to environmental change that occurs wtth agmg [1] may be partmlly due to a decrease m endocrine and neuroendocrme responsweness [2] A dechne in/3-adrenerglc car&ovascular responsweness has been demonstrated with age For example, aged rats [3] and humans [4] have a blunted car&ac chronotroplc response to lsoproterenoi Although the effect of agmg on acute, resting cardiac/3-adrenerglc responsweness has been studied, httle is known about the effect of aging on receptor adaptations to ongoing physiologic stress [5] Chromc hypoxla results m decreased/3-adrenerglc receptor (BAR) density m the hearts of Correspondence to David J Birnkrant, M D , Department of Pediatrics MetroHealth Medical Center 3395 Scranton R d , Cleveland, OH 44109, U S A

0047-6374/91/$03 50 Printed and Pubhshed m Ireland

© 1991 Elsevier Soentlfic Publishers Ireland Lid

136 young rats [6], perhaps due to increased levels of c~rculatmg catecholammes in the blood [6--8] There is, however, a report of unexpectedly increased BAR density in the lungs of rats exposed to chromc hypoxla [9] We speculated that aging might be accompanied by an impaired abdxty to adapt to stresses such as chronic hypoxla, which normally might require an increase m BAR density Therefore, we examined the ability of aged rats to adapt to chronic hypoxla with changes m lung BAR density MATER|ALS AND METHODS Chemtcals [125I]Iodocyanopmdolol (ICYP, spec act 2200 C1/mmol) was obtained from DuPont NEN Products (Boston, MA) B~ochemlcals were obtained from Sigma Chemical Co (St Louis, MO) Ammals Twenty-six young rats (aged 3 months) and 14 older rats (aged 20 months), all barrier-raised male Fischer-344 ammals, were obtained from Harlan Sprague Dawley breeders (Indianapolis, IN) Within 2 days of arrival, the rats in each age group were randomly dwlded into two groups The experimental group was placed m a temperature-controlled hypobanc hypoxic chamber (1/2 atm) for 3 weeks, in which 12 complete changes of air occurred hourly Normox~c control rats were housed in an identical chamber on the same hght-dark cycles at 1 atm pressure All rats had free access to food and water The protocol was approved by the Institutional Ammal Utlhzatlon Committee Membrane preparatton Rats were weighed, then anesthetized with pentobarbltal Blood was drawn for hematocnt determination, and the heart and lungs perfused with 20 cc cold buffered saline (20 mM Trls--HCI, 145 mM NaC1, pH 7 5) The lungs were removed, snap frozen In hquid nitrogen, and stored at - 7 0 ° C Lungs were later weighed, and thawed in 20 vols/g buffer A (250 mM sucrose, 5 mM TrIs--HCI, 5 mM EDTA, pH 7 4) This and all subsequent steps were performed at 4°C The lungs were minced, &srupted in a Polytron tissue grinder (Brmkmann Instrument, Westbury, NY) at setting 8 for 60 s, then homogenized with 10 strokes of a motor driven Teflon pestle (Wheaton Instruments, Mfllville, N J) Homogenates were passed through 4 layers of gauze and centrifuged at 270 x g for 10 mln The supernatant was disrupted by the Polytron at setting 7 for 60 s, then centrifuged at 47 800 x g for 15 mln The pellet was resuspended m buffer A, and centrifuged twice at 47 800 × g for 15 mln The final pellet was suspended in 6 vols/g buffer B (10 mM MgCI2, 50 mM Trls--HCI, pH 7 4) The membrane suspension was homogenized with 10 strokes in a hand-held homogenizer, atlquoted and stored at - 7 0 ° C until use

137

Bmdmg assays BAR denstty and antagonist affintty were determined by Scatchard analysts of binding isotherms for [125I]lodocyanoplndolol (ICYP) Protem (2 3 - - 9 8 #g) was incubated with 5--100 pM I C Y P in buffer B in a total volume of 300/zl for 110 mm at 37°C in the presence and absence of 10 -5 M propranolol to define non-specific binding Incubations were terminated by d1lution with 10 cc cold buffer B and bound ICYP was separated by raptd vacuum filtration through glass mlcrofiber filters (Whatman GF/C, Whatman, Clifton, N J) The filters were washed with 15 cc of buffer B, air drted and counted in a Hewlett-Packard g a m m a emission counter at 74% efficiency (Packard Instrument C o , Downer's Grove, IL) Stereoselectwlty of I C Y P b m d m g was determined by incubation of 50 pM I C Y P and 25/~g of protein wtth 10-3--10 -9 M (+) and ( - ) lsoproterenol

Stasttstwal analysis Groups were compared by 2-way A N O V A and unpaired Student's t-test Saturation isotherms were analyzed to determme K d for I C Y P and receptor concentration by the method of Scatchard [10] Stereoselectlvlty of I C Y P binding was determmed by comparmg IC50 for (+) and ( - ) tsoproterenol calculated by log-logtt analysis RESULTS

Phystologtc effects Both young and old rats exposed to 3 weeks of hypoxla weighed slgmficantly less than normoxlc rats (Fig 1) Hypoxtc rats also had a slgmficantly higher mean hematocnt (Fig 2) The body/lung weight ratio was increased with agmg and

500

400

E

300 200-

/Z 22

1000 Control

Yo%

/ 1

Hypoxlc Young n--13

Control Old n=7

Hypox=c Old n=7

Fig l Effectsofhypoxla and age on rat weight (mean ± S D ) Hypoxlc rats weighed less than controls (P < 0 001 by 2-way ANOVA), aged rats weighed more than young rats (P < 0 001)

1~8

75

P~ ,\1

K)

5O (a o

4)

'6

E Q' -7-

25

Control Young n=/

Hypoxic Young n=7

Control Old n=7

Hypoxlc Old n=7

h g 2 Elfe(-ts ot hypoxla and age on hemato(.r]t {mean 4- S D ) Hypoxlt. ral~ had a higher hemaloi.rit than controls ( P < 0 00l b) 2-~a'~ A N O V A )

_~,

,]

/~~~

1 500-

~ ooo-

m

/

e

# n --

•~• O~O A~A

0500-



o OOOo

^_^--A - -

z5

A--A-5b

Total Binding Specific Binding Non Specific Binding

--':

--A-

7's

~ A

16o

[ICYP], pM

® ~"

30

Q

r "~

20

is_

°

'i

0

100

200

300

400

500

Bound (fmol/mg) Fig 3 (a) S a m p l e s a t u r a u o n i s o t h e r m h y p o x ] c - y o u n g rat no s a t u r a t i o n isotherm Bin. x = 540 fmol/mg K d = 13 p M

15 (b) Scatchard I r a n s l o r m a t ] o n o| the

139

TABLE I EFFECTS OF HYPOXIA A N D AGE ON /3-ADRENERGIC RECEPTOR DENSITY Group

BAR denstty a * (mean ± S D ) (fmol/mg protein)

K d (pM)

Control-Young Hypoxlc-Young Control-Old Hypoxlc-OId

294 480 266 436

13 13 11 12

± ± ± ±

56 78 61 102

± ± ± ±

4 2 3 4

aNS-old vs young by 2-way ANOVA *P < 0 001-hypoxlc vs control by 2-way ANOVA

decreased with hypoxm ( c o n t r o l - y o u n g - 158 ± 56, hypoxlc-young - - 133 ± 21, control-old - - 201 ± 22, hypoxlc-old - - 158 ± 41) However, neither trend reached significance (P > 0 05 by 2-way ANOVA, old vs young, hypoxlc vs control, n = 4)

Ltgand bmdmg studtes Binding of ICYP was rapid, saturable and stereoselectlve with ( - ) lsoproterenol approximately 400 times more potent than (+) lsoproterenol (IC50 = 4 26 x 10-4M for (+) lsoproterenol, 1 × 10 -6 M for ( - ) isoproterenol) Non-specific binding was always < 10% of total binding Scatchard analysis yielded a straight line (r _ 0 90) consistent with a single class of antagonist binding s~tes (Figs 3a and b) BAR density was significantly higher in the lungs of hypoxlc compared to control rats Older and younger rats had similar lung BAR densmes Kd did not change significantly with hypoxla or aging (Table I) DISCUSSION

One of the concomitants of aging is impaired ablhty to respond to external stresses, and it has been suggested that this deficiency is due in part to reduced/3adrenerglc responsweness [2] For the lung, the literature generally suggests reduced /3-adrenerglc responsiveness with aging Such reduced responsiveness could be caused by decreased basal and stimulated adenylate cyclase activity, decreased receptor affinity for agonlst [l 1], and sequestration of receptors within the cells [12], all of which have been reported in the lungs of aged rats Although a reduction in BAR density could also contribute to reduced/3-adrenerg~c responsiveness, data are confllctmg on this point [13] and our own data show no significant difference in BAR densRy between young and old rats Hypoxm has been associated with Increased circulating catecholammes, decreased BAR density in rat hearts, and decreased basal and isoproterenol-stlmulated cardiac

140 adenylate cyclase activity as well as blunted cardiac chronotroplc response to infused lsoproterenol [6] However, in a single prior report, the lung BAR density was reported to be markedly increased by hypoxla [9] The effects of aging and hypoxla on the 3-adrenerglc system (especially the cardiovascular 3-adrenerglc system) have been well-studied separately [7], but the ability of the 3-adrenerglc system of aged ammals to adapt to chromc stress is not well understood We have previously presented evidence that elderly humans fall to upregulate lymphocyte BAR density appropriately in response to acute physiologic stimuli [5] The present study was undertaken to examine the ability of the 3-adrenerglc system of the lung to adapt to hypoxlc stress with aging, and particularly to examine the capacity for BAR upregulatlon during chromc stress We found increased lung BAR density in the lungs of both young and aged rats exposed to chronic hypoxla, consistent with Winter's report of increased BAR density in the lungs of young hypoxlc compared to normoxlc rats [9] We are confident that our rats experienced the hypoxlc stimulus, for hematocrlt was appropriately increased and weight decreased in exposed animals Increased BAR density with hypoxla is unexpected m light of the increased circulating catecholamlnes previously reported with hypoxla [8], and decreased BAR density in the hearts of rats exposed to chronic hypoxla [6] Yet the increase is large (63%) and consistent (httle overlap in BAR density between hypoxlc and normoxlc groups) Moreover, the increase in BAR density was comparable in young and old rats Thus, the capacity to upregulate BAR density in the lung In response to chronic stimulation is unimpaired in aged rats In Winter's study, there was a significantly higher proportion of 32 subtype adrenerglc receptors in the lungs of hypoxlc rats (79%) compared to controls (66%) Whde there is little data on rat lungs, quantitative radlohgand-bound BAR autoradlography studies in mice [14] and humans I15] have identified a high density of 32 subtype receptors in alveolar septae, with lesser densities in bronchlolar smooth muscle and vascular smooth muscle We found no dafference in body/lung weight ratio among groups, although there was a trend toward increased lung weight with hypoxla and decreased lung weight with aging Previous studies have described increased lung weight, thickened alveolar septae, an increase in thick-walled peripheral vessels [16], increased alveolar surface area [17] and increased volumes [18] in the lungs of rats exposed to chronic hypoxla The increased BAR density we found in lung homogenates from hypoxlc rats may therefore be due to vascular smooth muscle hyperplasla, perhaps associated with pulmonary artery hypertension, bronchlolar smooth muscle hypertrophy, or alveolar septal thickening - - all potentially maladaptlve responses Alternatively, the increased BAR density, predominantly 32 in subtype, may reflect increased alveolar surface area, associated with an increase in alveolar septae and type II pneumocytes, both of which contain a high concentration of 32 adrenerglc receptors Such a response would be adaptive, providing mcreased surface area for gas exchange

141 I n s u m m a r y , w e f o u n d a s i g n i f i c a n t i n c r e a s e in B A R d e n s i t y i n t h e l u n g s o f y o u n g and old rats exposed to chronic hypoxta crease lung BAR

Our data suggest that the capacity to m-

d e n s i t y m r e s p o n s e t o c h r o n i c h y p o x l a ts u n a f f e c t e d b y a g i n g

There was no significant difference in BAR density in the lungs of young compared to aged rats irrespective of hypoxic conditions

T h e a d a p t t v e s i g n i f i c a n c e o f t h e rtse

In B A R d e n s t t y i n r e s p o n s e t o h y p o x t a ts u n k n o w n ,

a n d ts d e p e n d e n t o n t h e s t a t e

o f a c t i v a t i o n o f t h e r e c e p t o r s i n v o l v e d a n d t h e p h y s i o l o g t c a l f u n c t i o n o f t h e cells o n which the receptors reside ACKNOWLEDGEMENTS We thank Beth Peticca ad Karen Totl for typing the manuscript

Thts study was

supported in part by grants HL-25830, HL-42215, and AG-00387 from the Nattonal Institutes of Health REFERENCES 1 J A Hems~mer and R J Lefkowltz, The impact of ageing on adrenerg~c receptor function chmcal and biochemical aspects J Am Gertatr Soc 33 (1985) 184---188 2 P J Scarpace. Decreased receptor actwatlon with age can it be explained by desensitization9 J Am Gertatr Soc, 36 (1988) 1067--1071 3 1 B Abrass, J L Davis and P J Scarpace, Isoproterenol responsweness and myocardaal #-adrenerglc receptors m young and old rats J Gerontol, 37 (1982) 156~160 4 E Lakatta, Age-related alterations In the car&ovascular response to adrenerg~c medmted stress Fed Proc 39 (1980) 3173--3177 5 S L Mader and P B Davis. Effect of age on acute regulatton of 13-adrenergsc responses m mononuclear leukocytes J Gerontol, 44 (1989) M168--173 6 N F Voelkel, L Hegstrand and J T Reeves et al, Effects of hypoxla on density of ~-adrenerglc receptors J Appl Phystol, 50 (1981)363--366 7 J -P Richalet, The heart and adrenerglc system m hypoxla In J R Sutton, G Coates, J E Remmers (eds), Hypoxla The Adaptanons, B C Decker, Inc, Toronto, 1990, pp 231--240 8 W L Cunnmgham, E J Becker and F Kreuzer, Catecholammes m plasma and urine at high altttude J Appl Phystol, 20 (1965)607--610 9 R J Winter, K E Dickinson, R M Rudd and P S Sever. Tissue speofic modulation of /3adrenoceptor number m rats with chromc hypoxm w~th an attenuated response to downregulat~on by salbutamol Chn Scl 70 (1986) 159--165 10 G Scatchard. The attraction of proteins for small molecules and ions 4nn N Y A~ud S~t 51 (1949) 660--672 11 P J Scarpace and I B Abrass, Decreased/3-adrenerglc agomst affimty and adenylate cyclase actlwty m senescent rat lung J Gerontol 38 (1983) 143--147 12 P J Scarpace and L A Baresl, Increased h'-adrenerglc receptors m the hght-denslty membrane fraction m lungs from senescent rats J Gerontol 43 (1988) B163--167 13 P Vanscheeuwljck, E Van de Velde and N Eraeymar, Effect of aging on properties and function of O-adrenoceptors m rat lung Eur J Pharmacol 172 (1989) 373--380 14 P J Henry, P J Rigby and R G Gol&e, Distribution of Bi- and B2-adrenoceptors In mouse trachea and lung a quantitative autoradlographlc study Br J Pharmatol, 99 (1990) 136~144 15 J R Carstalrs, A J Nlmmo and P J Barnes, Autora&ographlc vlsuahzat~on of/3-adrenoceptor subtypes m human lung Am Rev Resptr Dts, 132 (1985) 541--547

142

16 17 18

C Hunter, G R Barer J W Shaw and E J Clegg Growth of the heart and lungs m hypoxlc rodents a model of human hypoxlc disease (~hn S~t Mol Med 46 (1974) 375 ~91 D Bartlett Jr and J E Remmers Effects of high altitude exposure on the lungs ol young rats Resptr Phy~tol 13 (1971) 11~-125 P H Burn and E R Welbel, Morphometrtc e~tlmatlon of pulmonary diffusion capacity II Effect of PO 2 on the growing lung Adaptation of the growing rat lung to hypoxla and hyperoxla Rewtr Ph~,stol 11 (1971) 247 264

Chronic hypoxia increases beta-adrenergic receptor density in the lungs of young and old rats.

To test the hypothesis that the ability to regulate beta-adrenergic receptor (BAR) density in response to chronic hypoxic stress is impaired by aging,...
347KB Sizes 0 Downloads 0 Views