Effects of hypoxia lasting up to one month on the catecholamine content in rat carotid body

EFFECTS OF HYPOXIA LASTING UP TO ONE MONTH ON THE CATECH~LA~I~E CONTEXT IN RAT CAROTID BODY 1. MANBALIEK~, F. KARQWM~,S. WELLSTROM’and

S. LAHIR?

‘biochemical ~harmaco~o~ Section, Nationaf Heart. Lung, and Blood ~~st~t~~te~ Natronal Institutes of Health, Bethesda. Maryland 20205. U.S.A. ‘Laboratory of Clinic& Pharmacology, National Institute ol Mental Wrtalth. St. Elisabeth’s Hospital, Washington. DC 20032, USA. ‘Department of Physiology and Enstitute of Environmental Medicine, The University of Pennsylvania, Philadelphia. Pennsylvania, 19104. USA. Akgract-.-Long-term esposure to hypoxia (10~~~ O1 -!- 90yLN,) elicits an increase in the dopamine content of the carotid body after 2 days and in the noradrenaline content after one week. When hypoxia is extended to 3 weeks the dopamine and nor~drenaIin~ content increase IS- and f J-fold, respectively. These changes are associated with an increase in size and protein content of the carotid body. Removal %’ reduction of the naradrenaljne content. fails to ol the superior cervical ganglion, which causes a 5O!, prevent the increase in amine content elicited by hypoxia. Exposure to hypoxia for 4 weeks slightly increases the dopamine and 3,4-dihydroxyphenylacetic acid content in superior cervical ganglia and that of dopamine in adrena! glands, but fails to alter the levels of noradsenaline, adrenaline and 3-m&boxy-+ h~droxyph~ny~g~~col in heart and adrenal glands, that of noradrenaline in superior cervical ganglia aud that of dopamine and i.4-dihydroxgphenylacetic acid in the caudate nucleus. From these data it can be inferred that the increase in dopamine and noradrenaline content occurs as a specific response to long-term hypoxia and may involve bath induction of tyrosine hydroxylase and hyperplasia of glomus cells OFcarotid bodies.

LONG-TERMexposure to hypoxia elicits a number ol over a period of 99 days caused a 4.3-foId increase of homeostatic mechanisms which bring about changes the carotid body volume occupied by glomtls c& in circulation, in the rate of respiration and in puI- and sheath cells and a 16fold incttise in the volume mnnary gas exchange. In mammals the regulation of of capillaries, the acute response to hypoxia is triggered by stimuDespite the importance attributed to peripheral lation of chemoreceptors Located jn the carotid body. chemoreceptors in the regulation of hypoxia. little is Several laboratories have attempted to clarify whether known abaut the biochemical mechanisms operative the chemoreceptors in the carotid body are also trig in the carotid body during short- or long-term exgering the vent&tory changes during acclimatization posure to hypoxia. Substantial amounts of dopamine to prolonged hypoxia. It was shown that in absence of and noradrenaline have been found in the carotid carotid body chemoreceptors the hyperventilatory rebody of man and animals (C~ioccriro. BIKARDI & sponse during hypoxia is reduced (Brscn~~ Br VOGEL, TRAMEZZAX 1966; HELLSTROM.WAXBACER & CCSTA, 1971; BOWEROT & BVREAU, 1975; FORSTER, BKGARD 1976) and intravascular dopamine was shown to reduce the excitability of carotid sinus nci-ve endings & RASMUSSEN,1976) and ensuing h~~o~cnt~Iation and hypoxia may cause pulmonary hypertension (BISGARD (MITWELL & MCDONALD. 1975). Recent reports from & VOGEL, 1971). our laboratory showed that in rats the rxposurc to The &cts of’ prolonged hypoxia on rhe structure of hypoxia for 20 min decreases rhe dopamine content of the carotid body have been examined extensively in intact and denervated carotid bodies, while the norrats exposed to simulated high altitude in hypobaric adrenaline content remains unchanged (HAF*BRI..EH & chambers ~EDWAIKIS. 1911; k&Am, EDWARDS. WINHEWXROM, 1978). Studies on the turnover rate of SON & SMrnf, 1973). Morphometric studies showed dopamine in various experimental conditiuns showed that the average volume of the carotid body was that during short-term hypoxia the release rate ~2 about X6 times that of controls after exposure to 380 do~arn~~c increases, while its rate of synthesis &tits to torr for 5 weeka This increase in carotid body size change (HA~~~BAC:ER & HELLSTRC)#, 1978). Thus, these occurred in spite of a stunted overall body growth. results indicate: that the decrease of dopamine content F&%ING and WOLFF (1973) reported that hypoxia elicited by acute hypoxia is due to a release of this amine. A&tough studies on the long-term &kts or hypoxia have shown hypertrophy and hyperplasia of Ahhrr~iotiuns: DOPAC. j,4-dihydroxyph~nyl@lycol; HVA. homovanillic acid: MHPG, i-mrahtjxp$-hy&~xyvanious celluk~r structures in the carotid ho& phcnyIgIyc#i~ (EDWARDS, 1971: HEATII. EDWARDS, WPJSC,N &

82

1.

HANBAUER,F. KAROCIM. S. H~L.LSTKOM and S LAHIKI

SMITH, 1973; LAIDLER & KAY. 1978), the effects of prolonged hypoxia on the catecholamine content in carotid bodies have not yet been determined. The present experiments were carried out to study the levels of dopamine and noradrenaline in the carotid body of rats exposed to prolonged hypoxia. For comparison, the content of catecholamines and of their metabolites was studied in other structures of the central and peripheral nervous system. EXPERIMENTAL

PROCEDURES

Male Sprague-Dawley rats (Charles River: 150~200g) used in all experiments. The rats were fed Purina chow and were kept at 23°C with circadian illumination (14 h light). In one group of rats the superior cervical ganglion was removed unilaterally 7 days before exposure to hypoxia. Methohexital sodium (Brevitalm. Eli Lilly) was used to induce anesthesia. were

Long-term

exposure

to hyposiu

Several cages containing three rats each were placed in a chamber in which the gas composition was maintained at 10%02 + 90% N,. Groups of rats were kept in the chamber for various periods as indicated in ‘Results’. Assayfor

catecholamines

und

rheir metaholitev

Rats were killed by cervical dislocation and their carotid bodies, superior cervical ganglia, heart. adrenal glands. and caudate nucleus were rapidly dissected out. The various tissues were homogenized in appropriate volumes of 1 N HCI containing the appropriate deuterated standard for dopamine, noradrenaline, adrenaline. 3,4_dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). In brief. after centrifugation at 12,000 xg for 5 min aliquots of the clear supernatant were dried in microflex tubes. (Kontes Vineland, N.J.) under a stream of Nz. The residue of each tube was mixed with IO/d of ethyl acetate (dried over CaH,) and 100 ~1 of pentafluoropropionic anhydride (Pierce Chemical Co., Rockford, Ill.) and heated for IOmin at 70°C. The acylating agent was evaporated under Nz and the pentafluoropropionyl derivatives of dopamine. nor-

1000

0-o i -

adrenaline. and adrenaline were reconst~~ut~~din i 5 1~1 cthqi acetate. For the measurement of DOPAC ,~r-iJHVA. ?O” HC’I in methanol was added to anothe: X! i&fmccr,)tube? containing aliquots of the original supernatant :rnd mci+ bated at room temperature for IO mm. Tht: content of the tubes was dried under nitrogen. then 2.i ,il OI !(I”,, penra.. fluoropropionyl rmida7ole (Pierce (‘hemlial ( C.IIin ethki acetate were added and the tubes uerc iii-:tt~i .II 70 C‘ f ITrcpared ac dcscribed for the catecholamines. The 4000 Finnigan quadropole gaschrtmatograph-mass spectrometer was used. The mass to charge (m~‘c)ra!ios of the fragments selected for sin& ior! monitoring of dopamine. noradrcnatine, adrenaline. DOPAC. HVA and MHPG have heen described previousI> IKAROIM 6’: ‘41 1975: KAROLM& NEFF. 1979). The selectrci fragments \m MY of compound. m/e of deuterated isomer) were for dopamine (428 :43 I), noradrenaline (5%):592). adrenaline (604:607). DOPAC (387:392). HVA (283.X4) and MHPG (h2?:625).

RESULTS

Figure 1 shows the time course of the increase in the dopamine and noradrenaline content in the carotid bodies of rats exposed to hypoxi;t. An exposure lasting for 2 days increases the dopaminc content but fails to change that of noradrenaline. however an exposure lasting one week increases ths content of both dopamine and noradrenaline. Figure t shows that when the exposure time to hypoxia US extended to 2 and 4 weeks the dopamine and noradrenaline levels increase even further, After ;I 4 b\cek cxoosurt’ 1”

Dopomine Noradrenaline

weeks

ot exporvre

to hypoxia

FIG. I. Time-course of the changes in dopamine and noradrenaline content of rat carotid hod~c%chcited by prolonged exposure to 10% O2 in N,. The catecholamine content is measured mass-fragmentographitally and the data are expressed as p mol/pair carotid body +S.E.M.

Long-term hypoxia and carotid body catecholamines 1‘AnLfi

I,

INCREASE

fN

PROTEIN

AND

CATECHOLAMINE

CONTENT

kLICITI:D BY LONGTERM

.? days I week 7 weeks 4 weeks

CAROTID

BODY

1.22 2 0.15

3.3 * 0.46

f 2.2 21.2 ) 1.4 30.9 3: 3.3* 36.6 + 2.0** 56.7 + 4.x**

20.3

0

RAT

HYPOXIA

Noradrenaline Dopamine (nmol/mg protein + SEM)

Protein (Pg i sEM)

Time of exposure

IN

83

Il.3 & 1.7””

1.80* 0.23

II.3 1 1.X** IX.0 & 1.7**

1.73 & 0.27 3.83 k 0.46**

11.7* 2.0**

5.26& o.s7**

The protein concentration was determined by the method of LOWRY. RoSEBRouGH, & RANDALL. 1951,and the catecholamine content was measured mass-fragmentographically in one pair of carotid bodies FARR

hypoxia the diameter

of the carotid bodies is about 2.5 times larger than that of rats kept at room air.

This enlargement is associated with an increase in protein content. After I week the protein content increases by 52%. after 2 weeks by 801?/,after 4 weeks by 180% (Table 1). Table 1 also shows that the amount of dopamine expressed per units of protein increases after 2 days. while the amount of noradrenaline significantly increases only after 2 weeks of hypoxia. The

efSect of long-term

noradrenaline the superior

content cervicul

hypoxia

on the dopamine

of carotid bodies qj?er

and

removal

qf

ganglion

We have previously reported (HANBAUER & HELLthe removal of the superior cervical ganglion decreases the noradrenaline content of the carotid body by SOo/,. while the dopamine content remains unchanged. The data in Table 2 confirm this report and show that removal of the superior cervical ganglion fails to prevent the increase in the carotid body content of noradrenaline and dopamine elicited by an exposure to hypoxia for 16 days. Actually, the hypoxia-induced increase in the dopamine content is significantly larger in denervated than in intact carotid bodies. No significant difference could be noted in

STROM, 1978) that

TABLE 2. EFFECTOF LONG-TERMHYPOXlA ON TfIYT

IN CAROTID

BODY

AFTER

BILATERAL

the diameter of carotid bodies from intact and gangIioneclomized rats exposed to hypoxia for 16 days. qf long-term

E&t amines

und their

ior cervicul

hJyo.uia metaholites

ganglion,

heart

on the content in caudate and adrenul

tf catechol-

nucleus, glands

superof rats

In order to determine whether prolonged exposure to hypoxia increases the dopamine and noradrenaline content of carotid bodies selectively, we measured the content of various catecholamines and that of their metabolites in caudate nucleus, superior cervical ganglion, heart and adrenal glands. Table 3 shows that exposure to hypoxia for 4 weeks fails to alter the noradrenaline content in adrenal glands and in the superior cervical ganglion but significantly increases the dopamine content in these tissues. In the same rats the DOPAC content of superior cervical ganglia is increased, whereas the adrenaline DOPAC and MHPG content of adrenals fails to change. In rats exposed to hypoxia for 4 weeks the dopamine (0.13 k 0.014 n mol/mg), noradrenaline (4.3 + 0,23 n mol/mg) and MHPG (0.72 &-0.11 n moljmg) content in the heart and the dopamine (1.O + 0.23 n moljmg), DOPAC (0.37 + 0.034 n mol/mg) and HVA (0.22 5 0.028 n mol/mg) content of caudate nucleus remain unchanged.

THE

REMOVAL

DOPAMINE

AND

OF THE SUPERIOR

NORADRENALINE CERVICAL

CON-

GANGLION

-Condition

Dopamine Noradrenaline (pmol/pair cb i SEM)

Intact

59.6 + 5.0

27.2 i_ 3.0

Ganglionectomized

69.X ) 20.9

14.8 + 3.6t

Intact

‘28 + 32.0*

128 + 33.9*

Ganglionectomized

420 f 71.g*t

I51 & 34.9*

Room Air

Hypoxia II = 6; *p < 0.01 when compared with rats kept at room air. ip < 0.05 when compared with contralateral carotid bodies. Unilateral removal of the superior cervical ganglia was performed 7 days prior to exposure to hypoxia (10% O2 c 90% N,). Rats were exposed to hypoxia for I6 days. The dopamine and noradrenaline content was measured mass-fragmentographically.

IV RAT ADRENAL

(;LAND AND SUPERIOR ('FRVIC'AI GANC;LIA

Tissue

Adrenal Gland

Superior cervical ganglion

Dopamine ~oradrenaiin~ Adrenaline DOPAC MHPG -~^-~ Noradrenaline Dopamine DOPAC

i.9 -t 0 l0 ‘.I 1 t1.55 49.1)<: 2.2 0,029 .k O.iX~S 0.02x +: 0.002 -____-. .- .__~__._..“_ 7.1 :.. 0.17 O.ih t 0.019 0.12 t 0.c.m

I.4 _i !;.?il* 5s ;i IST.11Y :‘! 0.01X; il1)05 0.03; : ciiYH)h _. -.__.-._ 2.: G: -Y (),‘.J 1: __!i{j~~’ (I.?.:A “,(f#‘)p _” _.. ____

,I = 6: *p < 0.05. Rats were exposed to hypoxia (9%02 + 91% N2) for 4 weeks. The content of catecllolan~~urs and their metaholites was measured mass-fragmentographically.

In rats exposed to room air or to a mixture of 10% 02 in N, the ventilation was monitored plethysmographically at various periods in hypoxia, ne average ventilation at room air was 44ml x min-’ x lC@g-’ and this increased by 47% after I h, by 65% after I day and by 86% after 3 days of hypoxia (CARSON& LAHIRI, 1979). The ventilation remained elevated about 50% above control during l-4 weeks of hypoxia. DfSCUSSfON The present study indicates that long-lasting exposure to hypoxia increases the dopamine and noradrenaline content of rat carotid bodies. The increase in dopamine content elicited by exposure to hypoxia can be observed after 2 days in hypoxia, whereas an increase in noradrenaiine content can be measured after an exposure time of one week. Previous reports from our laboratory (HELLSTROM et a/., 1976; HANBAUER& HELLSTROM,1978) had shown that shortterm exposure to hypoxia decreases the dopamine content of carotid bodies without reducing the rate of dopamine synthesis, while it does not change the content nor the turnover rate of noradrenaline. These data indicate that different mechanisms are operative during short- and long-lasting hypoxia. Our previous work, which showed that acute hypoxia enhances the rate of release of dopamine, is in agreement with reports by BL~~MCKE, RODE& NEDORF (1967)and by HELLSTROM (1977). The tatter author demonstrated that the volume density of granulated vesicles in glomus cells decreases following ventilation of rats for 30 min with 5% O2 in N2. This reduction in volume density was interpreted to be due to a reduction in the number of synaptic vesicles because the size of the vesicles remained unchanged in hypoxia rats. Previous experiments also showed that exposure to hypoxia lasting at least 1 h increases the tyrosine hydroxylase activity of the carotid body after a latency time of 24 h (HANBAUER,LWENBERG& COSTA,1977). This change was due to an increase in the velocity

(Vmax) of the enzyme, which could be mcasurcd 24-48 h after exposure to hypoxia (HAXIIA~;ERcar~1.. 1977). The results of these studies lead to the conclusion that the initial increase in dopaminc and noradrenaline content of carotid bodies could be due to activation of tyrosine hydroxylase elicited by persistent hypoxia. From the observation that exposure to hypoxia for 4 weeks elicited an approx 2 3 fold enlargement of the size of the carotid body, it can be inferred that the persistent increase of the dopamino content caused by prolonged hypoxia may in part also be due to hypertrophy and hyperplasia of glomus cells. ~orphometric measurements bj LAnXf;a I% KAY (1975a,h) showed that after ?-I- 96 days of cxposure to hypoxia the number of glomus cells increased about 1.8 fold. Thus, their results indicate that hyperplasia itself accounts for less than 50”/;, of the increase in volume occupied by glomus cells and that the increase in cytoplasmic votumc of glomus ceils and in size of dense cored vesicles arc responsible for the larger part of the changes in dopamine content. The observation that removal of the qerior cervical ganglion augments the dopamine content of carotid bodies to a larger extent than in intact tissues leads to the assumption that noradrenergic nerve endings may exert a regulatory role on ciol?~mine-containing glomus cells. The enlargement of the vascr?lar bed of the carotid body. which receives its sympathetic Innervation from the superior cervical ganglion. has to he considered x being in part responsible for the inircase of the noradrenaline content in carotid bodies of rats exposed to hypoxia for several weeks. The lirst quantitative data on hypoxia-induced cellular modification of the carotid bodies reported by BLESSING& WOLFF (1973) showed that capillaries comprised lh”,,. glomus and sheath cells 56O/;,of the total volume. But in the enlarged carotid bodies of hypohii rats the cells formed 42% and the capillaries 3P”, of the volume. Since the present experiments show that removal of the superior cervical ganglion fails to prevent the Increase in noradrenaline content ol’ the carotid body elicited by hypoxia, it can be inferred that noradrena-

Long-term hypoxia

and carotid

85

body ~ate~holamines

The specificity of these changes in carotid body mechanisms is upheld by the finding that exposure to hypoxia for 4 weeks fails to change the catecholamine content in caudate nucleus, heart, superior cervical ganglia and adrenal glands. The slight increase in dopamine and DOPAC content in superior cervical ganglia and that of dopamine in the adrenal gland indicate that persistent hypoxia may cause a mild degree of metabolic stimulation also in these tissues. Studies on the regulation of tyrosine hydroxylase and the turnover-rate of catecholamines in these tissues are pidnned to clarify this point. However, the observed changes are small enough to be explained by the activation of local regulatory mechanisms,

line is also contained in glomus cells and that the number or the size of noradrenaline-containing glomus cells is also modified by chronic hypoxia. Moreover, the findings that the noradrenaline content which in carotid bodies of ganglionectomized rats is reduced by SOY,;),increases to the same level as in intact carotid bodies suggest that noradrenergic nerve endings may exert a regulatory control also on noradrenergic glomus cells and that the process of hypertrophy and/or hyperplasia of glomus cells is regulated either hormonaliy or by local regulatory factors. The increment in organ size and catecholamine content suggests an important role for the carotid body in cardiorespiratory regulation. In fact. it was suggested that an increased production and release of dopamine in glomus cells elicited by chronic hypoxia may decrease the ~ent~latory response to acute hypoxia (S~VERI~G~~A~S.BAINTOK & CARCTLEN. 1966).

Aclirro~.irdy4alrt1r-.-PartOFthis work was NIH Grants-NHLBI 19737-07 & OSX99-14.

supported

b!:

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