Brain Research, 579 (1992) 291-295 (~ 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

291

BRES 17693

Anemia up-regulates pontine A 1 adenosine receptors in fetal sheep Brian J. Koos a, Thomas F. Murray b and Waleed Doany a aDepartment of Obstetrics and Gynecology, Nicholas S. Assali Perinatal Research Laboratory, UCLA School of Medicine, Los Angeles, CA 90024 (USA) and bCollege of Pharmacy, Oregon State University, Corvallis, OR 97331 (USA) (Accepted 24 December 1991)

Key words: Brain; Chemoreceptor; Hypoxia; Respiration

A rise in central adenosine concentrations most likely triggers the inhibitory effects of acute hypoxia on fetal breathing movements. During prolonged O z deficiency, the incidence of fetal breathing increases over time, an adaptation which may involve down-regulation of adenosine receptors. Therefore, isovolemic anemia was induced in chronically catheterized fetal sheep to determine the effects of acute 0 2 deprivation on the affinity of brainstem m 1 adenosine receptors. Compared to control values, the mean hemoglobin concentration in 4 fetuses was lowered by 54% (to 3.7 + 0.4 g/dl) for 1 h and in 3 fetuses by 58% (to 3.8 + 0.6 g/dl) for 4 h. Mean preductal arterial pH during anemia was significantly reduced to values as low as 7.123 + 0.090, but mean paO 2 and paCO 2 were not significantly affected. The average incidence of fetal breathing movements was decreased by 80% during the first hour and by 50% during the fourth hour of anemia. In 3 fetuses with normal hemoglobin levels, about 32 __ 6.0% of the A 1 adenosine receptors in the brainstem were in the high affinity state. After 1 h of anemia, the percentage of high affinity receptors in the pons (but not medulla or midbrain) had significantly increased to 47 + 2.9%, but after 4 h of anemia all 3 brainstem regions had A 1 receptor affinity within the range of control values. It is concluded that acute anemia induces a short-lived up-regulation of pontine A~ adenosine receptors, but anemia does not alter A1 receptor coupling in the midbrain where A1 receptors related to breathing inhibition may be located. INTRODUCTION

t e r m i n e w h e t h e r this w o u l d desensitize A 1 a d e n o s i n e rec e p t o r s in the fetal b r a i n s t e m .

In s h e e p ( > 0 . 8 t e r m ) , fetal b r e a t h i n g m o v e m e n t s occur in e p i s o d e s a n d are p r e s e n t a b o u t 3 0 - 5 0 % o f t h e t i m e 7. A l t h o u g h h y p o x i a s t i m u l a t e s r e s p i r a t i o n p o s t n a tally, b r e a t h i n g activity in fetal s h e e p is a l m o s t c o m p l e t e l y e l i m i n a t e d by acutely r e d u c i n g fetal p a O 2 by 8 - 1 0 m m H g 3. This h y p o x i c i n h i b i t i o n of fetal b r e a t h i n g a p p e a r s to result f r o m t h e direct effects o f O z deficiency o n the fetal b r a i n s t e m 8'13'14, and r e c e n t e v i d e n c e indicates that the n e u r o m o d u l a t o r a d e n o s i n e helps m e d i a t e this r e s p i r a t o r y d e p r e s s i o n ~2, p r o b a b l y by stimulating m 1 a d e n o s i n e r e c e p t o r s 6'9'25. A n i n t e r e s t i n g f e a t u r e of h y p o x i c i n h i b i t i o n is the t r a n s i e n t n a t u r e o f this b r e a t h i n g d e p r e s s i o n . F o r e x a m ple, the i n c i d e n c e of fetal b r e a t h i n g m o v e m e n t s r e t u r n s to n o r m a l within h o u r s a f t e r t h e o n s e t of p r o l o n g e d 0 2 d e p r i v a t i o n 2'11'2°. A 1 a d e n o s i n e r e c e p t o r s exist in high and low agonist affinity states 23, and it is possible that the f u n c t i o n a l c o u p l i n g of A1 r e c e p t o r s to a r e l e v a n t efl e c t o r system m a y c h a n g e d u r i n g h y p o x i a , r e p r e s e n t i n g an a d a p t i v e r e s p o n s e which u n d e r l i e s the r e t u r n o f fetal b r e a t h i n g . B e c a u s e a n e m i a and h y p o x i a h a v e similar effects o n fetal b r e a t h i n g r e l a t i v e to c a l c u l a t e d b r a i n p O z 13, p r o l o n g e d a n e m i a was i n d u c e d in fetal s h e e p to h e l p de-

MATERIALS AND METHODS Ten pregnant ewes (Rambouillet-Columbia cross) were operated on under halothane anesthesia at 118-126 days' gestation (-0.8 term). Polyvinyl catheters (1.0 mm i.d.) were placed in a femoral artery and vein of the ewe. The fetal head was exteriorized, and polyvinyl catheters (0.5 mm i.d.) were inserted into a carotid artery, external jugular vein, the trachea and amniotic sac. The uterine and abdominal incisions were closed with suture, and the fetus and ewe were given at least 3 days to recover from surgery before starting experiments. Fetal tracheal pressure (minus amniotic fluid pressure) was recorded on a Grass polygraph (model 7E). Arterial blood gases and pH were measured using blood gas electrodes (Instrumentation Laboratories, model 1304). Hemoglobin concentration was determined with an OSM Hemoximeter (Radiometer, Copenhagen). After a 4 h recording of fetal breathing movements, isovolemic anemia was induced in the fetus by infusing intravenously (8 ml/ min) maternal plasma while simultaneously withdrawing fetal arterial blood at the same rate 15. When the fetal hematocrit had been reduced to 12-14%, fetal breathing movements were recorded for 1 (4 fetuses) or 4 (3 fetuses) h. Immediately after this study period, the ewe was euthanized (Eutha-6, Western Medical Supply), and the fetal brainstem was rapidly removed and dissected into the medulla, pons, and midbrain. The duration of anemia in these studies was based on our previous work in which fetal breathing activity was virtually abolished initially but returned to an incidence 50% of control values during the fourth hour of prolonged anemia 2°. In a control group (3 fetuses) breathing movements were recorded for

Correspondence: B.J. Koos, Department of Obstetrics and Gynecology, 22-132 CHS, Los Angeles, CA 90024, USA. Fax: (1) (213) 7949699.

292 4 h, and then brain tissue was collected as described. The brains of 3 newborn lambs (1-4 days old) were also evaluated for comparison with those in the control fetal group. Brain samples were quickly placed in separate plastic bags, rapidly frozen in liquid nitrogen, and stored at -70°C until analysis. A 1 adenosine receptor agonist binding 8-Cyclopentyl-l,3-[3H]dipropylxanthine ([3H]DPCPX) is an A Lselective antagonist radioligand which is not regulated by guanine nucleotides TM. The specific binding of [3H]DPCPX to brain A 1 adenosine receptors was determined in the presence of cyclopentyladenosine (CPA), an A 1 receptor agonist, to assess A t adenosine receptor agonist affinity states. The specific binding of [3H]DPCPX to fetal brain membranes was determined using a rapid filtration assay TM. For each brain region, 3-5 samples of brain tissue were analyzed per fetus. In each analysis, brain tissue (100 mg) was weighed and homogenized (Brinkman Polytron) in 100 vols. of ice-cold 50 mM Tris-HC1 buffer as described by Szot, Sanders and Murray z6. The homogenate was centrifuged (48,000 × g for 10 min) and the pellets were resuspended in the same vol. of ice-cold buffer and homogenized as before. The homogenate was recentrifuged, and the pellet was suspended in 100 vols. of 50 mM Tris-HC1 buffer containing 2 IU of adenosine deaminase (Sigma Type VI) per ml. After incubating for 30 min at 22°C, the homogenate was cooled on ice and recentrifuged. The pellet was resuspended in 100 vols. of ice-cold 50 mM Tris-HC1 with a Brinkman Polytron. Equilibrium binding reactions were performed in triplicate. In saturation binding studies, aliquots (0.175 ml) of the brain membrane suspension were incubated for 90 min at 22°C with [3H]DPCPX in 12 concentrations, varying from 0.04 to 1.9 nM. In competition binding experiments, aliquots of the membrane suspension were similarly incubated with 25 ,ul of 0.10.2 nM [3H]DPCPX (120 Ci/mmol) and 25/d of CPA (10 TM - 104), in a total vol. of 250/~1. Aliquots of the membrane suspension were also dissolved in 0.5 N NaOH, and protein concentration was determined 19. The equilibrium binding reactions were terminated by adding 2 ml of ice-cold Tris buffer, and the solution was filtered using Whatman GF/C filter strips and a Brandel Cell Harvester (model M-24 R, Brandel Instruments, Gaithersburg, MD). Non specific adsorption of ligand to filters was reduced by presoaking the filter strips in 0.5% polyethyleneamine. The filters were rinsed 4 times with 4 ml of ice-cold Tris buffer, and the filter disks were placed in vials with 3.5 ml of Biocount scintillation cocktail (RPI Corp., Mount Prospect, IL). A Beckman LS6800 scintillation counter was used to determine filter disk radioactivity. Total binding minus that present with l mM theophylline was designated as specific binding, which was approximately 98% of the total binding at the K d values for [3H]DPCPX. Analysis of data Saturation isotherm data were analyzed using the Lundon-1 iterative curve-fitting program (Lundon Software, Cleveland, OH). Agonist competition curves were analyzed using the iterative public procedure NEWFITSITES2 on the PROPHET system. The criterion for asserting a two-site model rather than a one-site model in these equilibrium competition analyses is the F statistic defined as follows: F = lSSl-SS2I/Idfrdfd

[SS2/df2] where SS~ and SS 2 are the sum of squares of the residuals for the one- and two-site fits, respectively, and dfl and df2 are the degrees of freedom for one- and two-site fits. Significant differences (P

Anemia up-regulates pontine A1 adenosine receptors in fetal sheep.

A rise in central adenosine concentrations most likely triggers the inhibitory effects of acute hypoxia on fetal breathing movements. During prolonged...
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