Hypoxia-induced changes in parasympathetic neurochemical markers in guinea pig heart

LINDA H. CROCKATT, DONALD D. LUND, PHILLIP G. SCHMID, AND ROBERT ROSKOSKI, JR. Cardiovascular Center, Departments of Biochemistry and Medicine and the Veterans Administration Hospital, University of Iowa, Iowa City, Iowa 52242; und Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 70112

CROCKATT,LINDA H., DONALD D. LUND, PHILLIPG. SCHMID, AND ROBERT ROSKOSKI, JR. Hypoxia-induced changes in parasympathetic neurochemical markers in guinea pig heart. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 50(5): 10174021, 1981.-Exposure of animals to hypoxia produces cardiovascular changes including right ventricular hypertrophy and alterations in heart rate. The activity of choline acetyltrans- ferase, a neurochemical marker of parasympathetic innervation, and the density of muscarinic choline@ receptors, measured by the binding of [3H]quinuclidinyl benzilate, were analyzed in the hearts of guinea pigs exposed to hypobaric hypoxia. We found a sign&ant increase in the activity of choline acetyl- transferase in the sinoatrial node in response to hypoxia after 7 or 14 days. In addition, we found significant decreases in the muscarinic receptor density in several contractile and conduct- ing regions. The decrease in receptor density may reflect regu- lation by the level of occupancy; an increased occupancy may produce a decrease in muscarinic receptor number.

parasympathetic innervation; muscarinic cholinergic receptor; choline acetyltransferase; acetylcholine

NEUROCHEMICAL ENZYME and receptor markers often respond to and serve as indices of altered neuronal activ- ity (21). Tyrosine hydroxylase activity, for example, in- creases in the peripheral sympathetic nervous system and adrenals in response to conditions that increase sympathetic nerve activity such as phenoxybenzamine injection (14) and cold exposure (29). However, assess- ments of choline acetyltransferase activity as a neuro- chemical marker of the parasympathetic nervous system have been much less extensive. This enzyme catalyzes the rate-limiting step in the biosynthesis of acetylcholine (19) and represents a marker of cholinergic innervation (14)

One goal of the present experiment was to test the premise that choline acetyltransferase activity serves as an index of parasympathetic nerve activity. Changes in nerve activity, moreover, may produce changes in recep- tor properties. There is considerable evidence for the reciprocal regulation of certain hormone and neurotrans- mitter receptors (13) by their corresponding effector. The P-adrenergic receptor number, for example, decreases in a variety of tissues in response to increased sympathetic stimulation by sympathomimetic drugs (20). There is a reciprocal increase in P-receptor number after chemical

sympathectomy following chronic guanethidine injection (II). Similarly, decreases in the number of insulin and glucagon receptors occur in hyperinsulinemia (10) and hyperglucagonemia (27), respectively. In each case, the response of the receptor is linked to the amount of neurotransmitter or hormone interacting with the recep- tor to produce an agonist-induced receptor decrease or, in the absence of agonist, a receptor increase.

After an initial tachycardia in humans and other mam- mals, hypoxia produces a bradycardia in the presence of increasing amounts of norepinephrine (17, 18). The par- ticipation of the sympathetic division of the autonomic nervous system in the heart rate response has been examined and is consistent with the idea that there is cardiac refractoriness to sympathetic stimulation (17). A second goal of the present study was to determine whether hypoxia may have an effect on the parasympa- thetic system using choline acetyltransferase activity and muscarinic receptor number as neurochemical markers.

After guinea pigs were exposed to hypobaric hypoxia for 7 and 14 days, the hearts were divided into specialized pacemaker and conduction tissue [sinoatrial (SA) and atrioventricular (AV) node, proximal bundle, and mod- erator band] and contractile tissue (atria and ventricles). We found an increase in choline acetyltransferase activity in the SA-nodal region, suggesting an increase in neu- ronal activity, neuronal mass, or a combination of these. In contrast to an increase in choline acetyltransferase activity, we found a reciprocal decrease in muscarinic receptor number in many heart regions.

METHODS

To produce hypobaric hypoxia, guinea pigs were placed together in a circular chamber (120 cm diam, 50 cm ht), producing a simulated altitude of 5,490 m, equivalent to 376 Torr pressure (23). Control animals were housed in a similar chamber at ambient pressure and temperature (23-25°C). Food and water were given ad libitum, and the chambers were cleaned daily, during which time the hypoxia animals were exposed to normal oxygen levels at atmospheric pressure for approximately 15 min. De- compression and recompression were performed during 90- and 5-min periods, respectively. Light and dark were alternated every 12 h.

Tissues were obtained from male (albino) guinea pigs

01.61-7567/81/oooO-oooO$Ol.25 Copyright 0 1981 the American Physiological Society 1017

1018 CROCKATT, LUND, SCHMID, AND ROSKOSKI

weighing 500-550 g. Hypoxia and control animals were chosen randomly and killed by cervical dislocation. The animals were killed 24 h after the final recompression and 15-90 min after the final decompression. Although heart rate and other physiological parameters change rapidly, others involving protein synthesis such as cardiac hypertrophy and polycythemia do not change during this relatively short time period. The specialized regions of the heart were dissected using landmarks specified by Anderson (2), thus allowing separate examination of the conducting tissues and contractile regions of the heart. After dissection, samples were immediately blotted, weighed, and stored in liquid nitrogen. Sampling was completed within 10 min of death; pilot studies showed that the muscarinic choline@ receptor and choline ace- tyltransferase activity were stable up to 45 min postmor- tem and for many months in liquid nitrogen.

The measurement of muscarinic choline@ receptor density in guinea pig heart was performed by a modifi- cation of the method of Galper and co-workers (9). Tissue was disrupted in 20 volumes of 5 mM potassium phos- phate (pH 7.4) with a Tekmar Tissuemizer (3 x 10-s bursts at a setting of 75, 4°C). Incubations were per- formed in Ringer’s solution (10 mM Tris l HCl, pH 7.4; 10 mM glucose; 150 mM NaCl; 3.5 mM KCl; 1.2 mM MgS04; and 1.2 mM NazHPOd) containing 1 nM C3H]quinuclidi- nyl benzilate ([3H]QNB) (13-17 Ci/mmol). By mass ac- tion, this concentration converts most of the receptor to the receptor-ligand complex and provides a measurement of the total number of receptors. In guinea pig heart, we found that at this concentration values are on the linear portion of Scatchard plots and that the ligand was com- pletely displaced by atropine or unlabeled QNB Over a few hours. Portions of tissue homogenate (0.25 mg tissue in 50 ~1) were added to the Ringer’s solution containing the [3H]QNB and incubated at 24OC for 45 min in dupli- cate. Nonspecific binding was determined in parallel assays in the presence of saturating concentrations (0.2 mM) of oxotremorine. Specific binding was that amount of binding inhibited by the oxotremorine and amounts to more than 90% of total binding. The reaction was termi- nated by filtration through a Whatman glass-fiber filter (GF/A) positioned over a vacuum. Unbound ligand was then washed through the filter with a saline wash (0.9% NaCl; 5 x 5 ml). Filters were dried for 10 min at lOO”C, and radioactivity was measured by liquid scintilIation spectrometry using Tritisol (8). Protein determinations of each sample were measured in duplicate according to the method of Lowry et al. (15).

Choline acetyltransferase activity was determined on the same tissue homogenates utilized for the t3H]QNB binding according to the method of Roskoski et al. (24). Statistical analyses were carried out using analysis of variance and Tukey’s test (28).

RESULTS

The characterization of the binding of [3H]QNB to rabbit (7) and chick (9) hearts has been described. Since there may be species-related differences in the specificity of binding of this compound, it was necessary to examine these parameters in guinea pig heart. Our studies indicate

that specific r3H]QNB binding was linear with tissue concentrations up to 1.5 mg/ml wet weight and that the binding was saturable when C3H]QNB concentrations were varied from 20 to 1,ooO pM. Scatchard analysis (26) yielded a linear plot with a dissociation constant (&) in the range of 50-120 pM, depending on the receptor concentration. Moreover, there was no indication of mul- tiple classes of binding sites or cooperativity. In addition, the specificity of [3H]QNB-receptor-complex formation was further substantiated by the displacement of r3H]- QNB with atropine, scopolamine, oxotremorine, and car- bamylcholine. Nicotine, on the other hand, was ineffec- tive in displacing [3H]QNB at concentrations up to 0.1 mM. Thus the binding of C3H]QNB in homogenates of guinea pig heart represents specific complex formation with the muscarinic-cholinergic receptor. This is in agree- ment with the observations of other investigators for a variety of species and tissues (7,9).

The effect of 7 and 14 days of exposure to hypobaric hypoxia on parasympathetic neurochemical markers in guinea pig heart was determined. After 7 days of expo- sure, a 55% increase in choline acetyltransferase activity occurred in the SA-nodal region, and a 34% increase occurred in the interventricular septum (Table 1). After 14 days of exposure, choline acetyltransferase activity in the region of the SA node may reflect increased parasym- pathetic nerve action in pacemaker tissue and may rep- resent a neurochemical correlate of reflex activation of the parasympathetic system in response to hypobaric hypoxia.

No change in the apparent Km of the enzyme for acetyl- CoA, determined as previously described (25), was ob- served in the experimental groups. Changes in the num- ber of enzyme molecules, not changes in substrate affm- ity, appear to account for the observed increase in en- zyme activity. The activities of combined experimental and control homogenates were additive; this makes the presence of an activator in the homogenates of the ex- perimental group unlikely (not shown).

The amount of the second neurochemical marker, the muscarinic receptor, was measured in the same homog- enates by C3H]QNB binding. After 7 days, significant (P < 0.05) decreases in the amount of receptor occurred in the right atrial appendage and right ventricle (Table 2). After 14 days, the number of receptors decreased in the SA and AV nodes, right atrial appendage, and right

TABLE 1. Choline acetyltransferase activity in heart after hypobaric hypoxia

Region COIltTOl 7-Day Hypoxia

14-Day Hypoxia

Right ventricle 56.2 k 12 48.1 A 6.4 48.7 A 7.0 Right atrial appendage 80.4 k 13 91.2 * 9*9 77.9 k x5 SA node 93.3 t 14 143 * 14* 159 r+, 14* AV nude 110 * 22 125 z!z 13 105 zt 16 Left atrial appendage 47.9 * 5.7 50.7 * 9.4 54,s z!L 9.7 Posterior left ventricle 80,4 zk 13 91.2 z!z 10.2 78.0 k 7.2 Interventricular septum 43.3 k 4.2 58.1 k 4.1* 52.1 * 7.3

Values are means k SE. Choline acetyltransferase activity (nmo1.g tissue-’ g h-‘) deter& mations performed in duplicate as previously de- scribed (25). Analysis of variance and Tukey’s test used to determhe statistical significance (28). * P < 0.05 compared with control.

HYPOXIA AND HEART 1019

ventricle (P < 0.05). The distribution of receptor changes was more widespread than that of the changes in choline acetyltransferase activity.

To determine whether the decrease represented changes in receptor number or a change in ligand affinity, Scatchard analysis of [“HIQNB binding was performed. Since the receptor concentrations were greater than 0.1 &, analyses were performed at equal receptor concen- trations to allow direct comparison of the dissociation constants and receptor densities for control and hypoxia- exposure samples (3). In response to hypobaric hypoxia, the SA-nodal region exhibited a 50% decrease in receptor density and no significant change in apparent & (Fig. I) as determined by Scatchard analysis. Similar analyses were performed on each sample in Table 2 where signifi- cant differences occur. In every instance, the differences reflect changes in receptor number; no changes in appar- ent & were observed. Scatchard analyses were also performed on regions not exhibiting changes in receptor density. No changes in & or receptor number were found (not shown).

To examine the possibility that an inhibitor that caused a reduction in radioligand binding was present in the hypoxia-exposure samples, reconstitution experi- ments were performed. [3H]QNB binding was deter- mined in hypoxia and control heart-sample homogenates

TABLE 3. r3H/QNB binding in reconstituted hypoxia and control heart humugenates

[“H]QNB Binding, fmol l mg protein-’

Control 92 k 8 Hypoxia 54 2 8 Combined 137 4.9

Values are means -t SE; rz = 4 animals per group. Binding measured on 25-~1 portions of control or hypoxia (7-day) heart homogenates alone or in combination as described in METHODS.

TABLE 4. Neuruchemical markers in lung in response to hypo baric hypoxia

Choline Acetyltransferase Activity, nmol l g tissue- ’ l

E3H]QNB Binding, fmol .

h-’ mg protein-’

Control l2,0 zk 2.2 62.1 k 9.0 Hypoxia 12.8 t 1.3 53.8 zk 8.4

Values are means t, SE; n = 6 animals per group. Choline acetyl- transferase activity and [3H]QNB binding assays performed on lung homogenates obtained from animals exposed to hypobtic hypoxia (7- days) or controls as documented in METHODS.

TABLE 2. 13H/QNB binding in keart after hypo baric hypoxia

alone or in combination. The binding was additive (Table 3). This suggests that no inhibitor was present in the homogenates derived from the hypoxic animals.

To investigate whether the changes in enzyme activity and receptor density following exposure to hypobaric hypoxia were a result of decreased oxygen, rather than the reduced atmospheric pressure, we examined [3H]- QNB binding and choline acetyltransferase activity in the hearts of guinea pigs exposed to hypoxia for 7 days at normal atmospheric pressure (normobaric hypoxia). One group of six guinea pigs was housed in a chamber fitted with hoses leading to Na and compressed air. The gases were mixed before entering the chamber, and the flow rates were adjusted so that the chamber contained 8% Og-92% Nz and was completely reventilated every 10 min; 02 was monitored periodically with a Beckman oxygen analyzer. A second group of guinea pigs was housed in a chamber connected to compressed air; distal vents en- sured maintenance of atmospheric pressure. The results agreed with those given in Tables I and 2 for the 7 days of exposure to hypobaric hypoxia. These experiments are consistent with the notion that the alteration in neuro- chemical parameters are the result of hypoxia and not pressure.

As these studies progressed, it became evident that hypoxia altered the parasympathetic neurochemical pa- rameters of the heart. To test the specificity of the response, other organs were examined for their levels of choline acetyltransferase activity and muscarinic recep- tor during exposure to hypoxia. Among these, kidney and spleen from guinea pigs with hypoxia for one week were assayed for [“HIQNB binding and enzyme activity. The assays were performed as described for heart in METH-

ODS. No significant level of activity was found in the control or hypoxia groups, indicating little parasympa- thetic innervation. The guinea pig lung, however, ex- hibited measurable activity. The values from experimen- tal samples were not statistically different from those of the control group (Table 4). Although the lung exhibits

Region Control 7-Day Hypoxia

14-Day Hypoxia

Right ventricle 205 k 11 157 k If* 137 t 14* Right atrial appendage 178 k 14 135 k 12* 123 t, 9* SA node 179 k 27 120 k 14t 83 t 16” AV node 141 t, 30 160 k 27 49 * 2” Left atrial appendage 181 k 14 192 k 18 176 k 21 Posterior left ventricle 103 -t- 27 144 k 8 141 * 6 Interventricular septum 177 zk 29 108s9t 117 & lot

Values are means k SE; n. = 5 animals per group. r3H]binding activity (fmoL mg protein-‘) determined on same samples presented in Table 1 as described in METHODS. * P < 0.05 compared with control. + P < 0.10 compared with control.

0.4

8/F 0.2

I 1 I I

10 20 30 40

Bound (fmol)

FIG. 1. Scatchard plot derived from specific binding of concentra- tions of E3H]QNB ranging between 20 and 1,000 pM, A, SA node from guinea pigs after 14 days hypobaric hypoxia; a, SA node from control guinea pigs. Apparent &‘s, 90 nM, Amount of receptor, 186 k 16 and 92 * 11 fmolgmg protein-’ for control and hypoxia samples, respec- tively. Assays performed as described in text using 0.30 and 0.50 mg protein l ml-’ for control and hypoxia samples, respectively.

1020 CROCKATT, LUND, SCHMID, AND ROSKOSKI

marked physiological changes in response to hypoxia (i.e., constriction of the vasculature), no changes were evident in the parasympathetic neural parameters. This observation and the finding that not all heart regions exhibit changes in response to hypoxia provide evidence for the specificity of the effect on the parasympathetic nervous system of the heart.

DISCUSSION

The SA node, th .e primary pacemaker of the heart, is densely innervated by parasympathetic nerve fibers (1). The increase of choline acetyltransferase activity in this region after 7 and 14 days of exposure to hypoxia suggests that there is increased parasympathetic nerve activity in heart. The increase in enzyme activity may result from two processes 1) an increase in existing nerve terminals, or 2) a proliferation of cholinergic nerve endings. Our previous studies suggest that vagal nerve proliferation associated with sprouting occurs within 48 h (16).

Changes in choline acetyltransferase activity have been observed in other experiments. For example, reflex activation of the parasympathetic system to salivary glands resulted in an increase in enzyme activity (5). In response to physical training in rats, an increase in atrial, but not ventricular, transferase activity occurred (6). These results support the concept that increased para- sympathetic activity is associated with bradycardia in physical training.

Changes in heart rate have been demonstrated in humans, dogs, and goats in response to hypobaric hy- poxia (12, 16, 17). Hartley et al. (12) reported that in- creases in heart rate follow atropine injection in exercis- ing humans at altitude (4,600 m) but not at sea level. They also suggest that parasympathetic activity is greater at high altitude. Maher et al. (17), on the other hand, found no differences in resting heart rate in goats at altitude (4,300 m) before or after methylatropine in- jection; no evidence for increased vagal activity was found in the latter experiments. Whether this is related to species differences or other parameters requires fur- ther study. Because of technical difficulties, we have been unable to obtain reliable heart rates in resting unanesthetized guinea pig. The increase in choline ace-

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tyltransferase and the decrease in the amount of musca- rinic receptor suggest that there is an increase in para- sympathetic activity in guinea pigs during hypobaric hypoxia.

In coptrast to increased choline acetyltransferase ac- tivity, the cardiac muscarinic receptor number decreases in specialized pacemaker and conducting tissue and con- tractile tissue. The receptor decrease is consistent with the notion of increased parasympathetic activity in re- sponse to hypoxia. The regulation of a receptor by its occupancy is common in hormone or neurotransmitter receptor-mediated processes (10, 11). Few examples of muscarinic-receptor regulation, however, are known. In cardiac tissue, for example, the regulation of the musca- rinic receptor by nerve activity has been shown in a collaborative study. Roeske et al. (22) have demonstrated an increase in muscarinic cholinergic receptors in extrins- ically denervated (transplanted) rat hearts. Transplan- tation severs all extrinsic nerves to the heart, thereby abolishing all preganglionic parasympathetic and post- ganglionic sympathetic neural input to the heart. In response to denervation we find an increased muscarinic receptor number (22).

A comparison of enzyme and receptor responses to hypoxia (Tables 1 and 2) reveals that changes in [“HI- QNB binding are more widespread than changes in en- zyme activity levels. This may simply be accountable by a difference in the sensitivity of response of the two neurochemical parameters; perhaps [3H]QNB binding is a more sensitive index of change for parasympathetic nerve activity. More experimentation is required to con- firm this idea. With hypoxia, it may be possible that there are enough increases in nerve activity (traffic) to mediate receptor decreases, but not enough to increase choline acetyltransferase activity. At present, the degree of change of parasympathetic activity required to pri>- duce receptor decreases is unknown; other parameters such as the acetylcholine turnover or vagus recording may provide this information.

This work HL- 14388.

was supported by National Institutes of Health Grant

Received 21 May 1980; accepted in final form 15 December 1980.

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