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A ²H-NMR study of lipid chain disorder in a model membrane : effect of integral peptide length Macqueen, Robin Michael 1986

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THE EFFECTS OF PROLONGED STATIC INFLATION ON THE DISCHARGE CHARACTERISTICS OF PULMONARY STRETCH RECEPTORS IN TURTLES by HEATHER ANN MCLEAN B.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF ZOOLOGY) We accept t h i s t t f e s i s as conforming to the required,, sta^da^d THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1986 © H.A. McLean In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Zoology The U n i v e r s i t y of B r i t i s h Columbia Vancouver, B.C. V6T 2A9, Canada Date i ABSTRACT Both the t o n i c and p h a s i c d i s c h a r g e c h a r a c t e r i s t i c s o f s i n g l e s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s (SARs) were examined b e f o r e and a f t e r a one hour p e r i o d o f m a i n t a i n e d l u n g volume i n f r e s h w a t e r t u r t l e s , Pseudemvs s c r i p t a and Chrysemys p i c t a . Lung volume was m a i n t a i n e d a t e i t h e r r e s t i n g volume ( a i r w a y p r e s s u r e = 0 cmH20) or a t an e l e v a t e d l u n g volume ( a i r w a y p r e s s u r e = 10 cmH20). P r e s s u r e i n f l a t i o n s were performed w i t h both a i r and 5% C02 i n a i r . Two p o p u l a t i o n s o f r e c e p t o r s were r e c o r d e d from: low t h r e s h o l d SARs ( t h o s e t h a t e x h i b i t e d t o n i c d i s c h a r g e a t r e s t i n g l u n g volume) and h i g h t h r e s h o l d SARs ( t h o s e t h a t d i d not e x h i b i t t o n i c d i s c h a r g e a t r e s t i n g l u n g volume). D u r i n g the one hour p e r i o d o f m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r , low and h i g h t h r e s h o l d SARs adapted up to 80% and 30% r e s p e c t i v e l y . F o l l o w i n g t h i s p e r i o d , the peak d i s c h a r g e r a t e a t t a i n e d w i t h s t e p i n f l a t i o n was unchanged i n both groups. The low t h r e s h o l d SARs demonstrated a d e c r e a s e i n the p h a s i c component o f d i s c h a r g e a s s o c i a t e d w i t h dynamic l u n g i n f l a t i o n s f o l l o w i n g one hour o f m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r but the h i g h t h r e s h o l d r e c e p t o r s d i d n o t . D u r i n g one hour o f m a i n t a i n e d l u n g i n f l a t i o n w i t h 5% C02 i n a i r , low and h i g h t h r e s h o l d SARs adapted more t h a n they d i d d u r i n g m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r . F u r t h e r m o r e , t h e r e was an o v e r a l l d e c r e a s e i n both the peak d i s c h a r g e a t t a i n e d w i t h s t a t i c l u n g i n f l a t i o n and the p h a s i c r e s p o n s e s to dynamic l u n g i i i n f a l t i o n s following maintained lung i n f l a t i o n with 5% C02 i n both groups of r e c e p t o r s . During the one hour p e r i o d o f maintained lung i n f l a t i o n with a i r , t here were s i g n i f i c a n t i n c r e a s e s i n both lung gas and blood gas l e v e l s of C02, decreases i n a r t e r i a l pH and decreases i n lung gas oxygen l e v e l s . The decreases i n t o n i c and phasic components of SAR discharge seen during the one hour p e r i o d of maintained lung i n f l a t i o n with a i r are due i n p a r t to an accumulation o f metabolic (lung gas) C02. i i i TABLE OF CONTENTS Page A b s t r a c t i L i s t o f T a b l e s i v L i s t o f F i g u r e s v Ack.nowlegdements v i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods 22 R e s u l t s 32 D i s c u s s i o n 58 L i t e r a t u r e C i t e d 88 iv L i s t of Tables Page Table 1 Blood and Lung Gas Measurements 51 V L i s t of Figures Page Figure 1 Outline of Nerve Recording Protocol 26 Figure 2 Outline of Blood and Lung Gas Protocol 29 Figure 3 Response of SAR #11 to S t a t i c Lung I n f l a t i o n and Deflation 33 Figure 4 Response of SAR #11 to Dynamic Lung Infl a t i o n s . 34 Figure 5 Low Threshold Fibers; Response to Maintained S t a t i c Lung Inf l a t i o n s 36 Figure 6 Adaptation Indices for Low Threshold Slowly Adapting Pulmonary Stretch Receptors 39 Figure 7 Low Threshold Fibers; Response to Dynamic Lung Inf l a t i o n s (Discharge Rate) 41 Figure 8 Low Threshold Fibers; Response to Dynamic Lung Inf l a t i o n s (Impulses) 43 Figure 9 High Threshold Fibers; Response to Maintained S t a t i c Lung In f l a t i o n s 45 Figure 10 High Threshold Fibers; Response to Dynamic Lung In f l a t i o n s (Discharge Rate) 47 Figure 11 Lung Gas Analysis - Air 50 Figure 12 Blood Gas Analysis - Air 52 Figure 13 Lung Gas Analysis - 5% C02 54 Figure 14 Blood Gas Analysis - 5% C02 55 Figure 15 Response of a Rapidly Adapting Pulmonary Stretch Receptor 57 v i Page F i g u r e 16 C o n t r i b u t i o n of C02 to Low Threshold Pulmonary S t r e t c h Receptor A d a p t a t i o n 76 F i g u r e 17 C o n t r i b u t i o n of C02 to High Threshold Pulmonary S t r e t c h Receptor A d a p t a t i o n 79 Fig u r e 18 SAR Discharge vs Lung Volume i n a H y p o t h e t i c a l 500 gram T u r t l e 82 v i i ACKNOWLEGDEMENTS F i r s t and foremost I would l i k e to express my g r a t i f i c a t i o n to Drs. W.K. Milsom and G.S. M i t c h e l l f o r allowing me to become a p a r t o f t h e i r c o l l a b o r a t i v e study which i n t u r n became the foundation of t h i s t h e s i s . I would a l s o l i k e to thank Dr. M i t c h e l l f o r his support, guidance and f r i e n d s h i p which have continued long a f t e r the completion o f the experiments. I wish to thank Dr. Milsom f o r his endless humor, patience, teaching and fri e n d s h i p . I'd l i k e to thank my family f o r t h e i r undying support and comfort. F i n a l l y , I wish to express by si n c e r e a p p r e c i a t i o n to a l l of the members of the Milsom Lab. Marianne, S a l l y , C h e r y l , Graham, Greg and Harv; thankyou so much f o r your laughter, your help and your encouragement. I'd be s u r p r i s e d i f any of us ever found another lab l i k e t h i s ... but then there's only one Captain Zot!! 1 INTRODUCTION The function of breathing i s to ensure that there i s an adequate delivery of oxygen to, and removal of carbon dioxide from the gas exchange surface. The r e s p i r a t o r y system, in conjunction with the cardiovascular system, provide the cel l s with the oxygen required f o r processes v i t a l to their metabolism. In addition, these systems e s t a b l i s h a means by which carbon dioxide, the primary metabolite of oxidative reactions, i s removed from the body. The o v e r a l l l e v e l of minute ve n t i l a t o n is act i v e l y adjusted through a number of c o n t r o l mechanisms to maintain adequate exchange over a wide range of metabolic states. These c o n t r o l mechanisms also serve to es t a b l i s h the breathing pattern. The amount of ai r moved i n and out of the lungs each minute (minute ventilation, V (ml/min)) i s the product of t i d a l volume (VT (mis)) and breathing frequency (min -1). The frequency component of V is i n turn a function of the ins p i r a t o r y i n t e r v a l (TI) and the expiratory i n t e r v a l (TE). The r e l a t i v e levels of VT, TI and TE es t a b l i s h a breathing p a t t e r n (Bradley, 1977). CONTROL OF VENTILATION: The o v e r a l l l e v e l of minute v e n t i l a t i o n i s controlled p a r t i a l l y through a f f e r e n t information a r i s i n g from the peripheral and c e n t r a l chemoreceptors (Mitchell, 1980). In mammals there are two populations of peripheral chemoreceptors: one located a t the b i f u r c a t i o n of the common c a r o t i d a r t e r y (carotid body chemoreceptors) and the other located near the 2 a o r t i c arch (Daly, 1983). The chemoreceptors are d i s t i n c t not only in their anatomical location, but also in their responses to r e s p i r a t o r y stimuli. Both c a r o t i d and a o r t i c body chemoreceptors readily respond to steady-state decreases in a r t e r i a l blood oxygen tension by increasing their discharge. However, steady-state increases in a r t e r i a l blood carbon dioxide tension and/or decreases in a r t e r i a l pH e l i c i t a greater increase in discharge from the c a r o t i d body chemoreceptors than from the a o r t i c body chemoreceptors (Lahiri e_ al., 1983). C e n t r a l chemoreceptors are s i t u a t e d near the v e n t r o l a t e r a l surface of the medulla (Loeschcke, 1983) and respond to changes in the carbon dioxide tension of the e x t r a c e l l u l a r f l u i d (ECF) in the brain. Molecular carbon dioxide (C02) f r e e l y d i f f u s e s across the blood-brain b a r r i e r to the ECF, is readily hydrolyzed and di s s o c i a t e s to produce hydrogen ions (H+) and bicarbonate ions (HC03-) (Slonim & Hamilton, 1971). The s p e c i f i c stimulus for the c e n t r a l chemoreceptors is thought to be the increased concentration of H+ ions near the area of the receptors (Loeschcke, 198 3). The r e f l e x response from the stimulation of both the c e n t r a l and peripheral chemoreceptors is manifest through a change in the o v e r a l l l e v e l of v e n t i l a t i o n as well as concomitant changes in breathing p a t t e r n (Mitchell, 1980). CONTROL OF THE BREATHING PATTERN: Air-breathing v e r t e b r a t e s demonstrate two types of breathing patterns, rhythmic and arrhythmic. Under normal physiological conditions, rhythmic breathing patterns are seen in f i s h , birds 3 and mammals while arrhythmic patterns are c h a r a c t e r i s t i c of most r e p t i l e s and some amphibians. The majority of the studies pertaining to the generation and maintenance of the breathing p a t t e r n have been conducted on mammals. Generation of r e s p i r a t o r y rhythm is a complex phenomenon involving extensive contributions from many motor neuron pools (for review see Euler, 1986). The primary r e s p i r a t o r y rhythm generators are sit u a t e d in the medulla and the pons. In the medulla, there are a t l e a s t two d i s t i n c t r e s p i r a t o r y neuron groups closely associated with the c e n t r a l r e s p i r a t o r y rhythm generator: the v e n t r a l r e s p i r a t o r y group (VRG) located near the nucleus retroambigualis and the nucleus para-ambigualis and the do r s a l r e s p i r a t o r y group (DRG) located near the nucleus t r a c t u s s o l i t a r i u s (Euler, 1986). The DRG contains primarily i n s p i r a t o r y neurons whereas both in s p i r a t o r y and expiratory neurons are found in the VRG (Mithcell, 1980). The pontine region has two re s p i r a t o r y neuron pools (the nucleus parabrachialis medialis and the Kolliker Fuse nucleus) which are not involved i n the generation of r e s p i r a t o r y rhythm per se. but are instrumental in influencing the timing of i n s p i r a t i o n and expiration. P a t t e r n generating neurons are also found in the phrenic motor nucleus of the spinal cord. This spinal rhythm generator i s capable of maintaining r e s p i r a t o r y rhythm in the absence of higher centers but i n the i n t a c t animal, the c e n t r a l rhythm generators are of primary importance (Mitchell, 1980). The rhythm produced by the c e n t r a l p a t t e r n generators i s 4 modified by a number of f a c t o r s . Parasympathetic a f f e r e n t fibers of the tenth c r a n i a l (vagus) nerve projecting from the pulmonary receptors to the d o r s a l r e s p i r a t o r y group located near the nucleus t r a c t u s s o l i t a r i u s are important in the c o n t r o l of r e s p i r a t i o n (for review see Sant'Ambrogio, 1982). From here, the DRG sends fib e r s to the phrenic motor nucleus in the v e n t r a l horns of the spinal cord ( c e r v i c a l segments 3,4, and 5), the r e s p i r a t o r y airways, as well as r o s t r a l l y to i n s p i r a t o r y and expiratory neuronal pools within the v e n t r a l r e s p i r a t o r y group. From the i n s p i r a t o r y and expiratory neurons in the VRG, e f f e r e n t axons extend c o n t r a l a t e r a l ^ to the i n t e r c o s t a l and the phrenic motor neuron pools (Mitchell, 1980). Pulmonary Receptor C h a r a c t e r i s t i c s Maintenance of the normal breathing pat t e r n is largely dependent upon a f f e r e n t information a r i s i n g from pulmonary receptors. There are three basic types of pulmonary receptors; slowly adapting pulmonary s t r e t c h receptors, rapidly adapting pulmonary s t r e t c h receptors and juxta-pulmonary cap i l l a r y receptors (for review see Paintal, 1977). Rapidly adapting pulmonary s t r e t c h receptors (RARs) were f i r s t described by Larrabee and Knowlton (1946). RARs are located exclusively in the larger diameter airways O0.3 mm) and respond to large changes in transmural pressure. Unlike slowly adapting pulmonary s t r e t c h receptors, these mechanoreceptors adapt quickly and completely to a maintained stimulus (Paintal, 1973). Rapidly adapting pulmonary s t r e t c h receptors are also 5 stimulated by many noxious inhalents and are therefore commonly r e f e r r e d to as i r r i t a n t receptors. Ammonia, aerosols and cig a r e t t e smoke are p a r t i c u l a r l y e f f e c t i v e in stimulating RARs. The t y p i c a l r e f l e x e l i c i t e d by the stimultion of RARs is the cough r e f l e x as well as an increase in minute v e n t i l a t i o n (Paintal, 1977). Juxta-pulmonary cap i l l a r y receptors (J-receptors) are mechanoreceptors which are located in the i n t e r s t i t i a l lining of the lungs close to the pulmonary cap i l l a r y bed (Paintal, 1969). The n a t u r a l stimulus for these receptors i s an increase in i n t e r s t i t i a l volume. Any condition (pathological or otherwise) that leads to pulmonary edema may r e s u l t in the stimulation of J-receptors. Vigorous exercise frequently produces pulmonary edema and i t has been postulated (Paintal, 1970) that stimulation of J-receptors may r e s u l t i n termination (or at l e a s t reduction) of exercise through c e n t r a l , r e f l e x inhibition of the limb muscles. However, there i s not much evidence to support this postulated r e f l e x and the physiological significance of J-receptor stimulation remains a c o n t r o v e r s i a l issue. Both rapidly adapting pulmonary s t r e t c h receptors and juxta-pulmonary cap i l l a r y receptors are only a c t i v a t e d under peculiar circumstances. Neither the RARs nor the J-receptors are involved in establishing the normal breathing pattern. Unless otherwise stated, a l l further discussion of pulmonary receptors and their contribution to the c o n t r o l of the breathing p a t t e r n will p ertain only to slowly adapting pulmonary s t r e t c h receptors 6 since these are the main contributors to breathing p a t t e r n c o n t r o l under most conditions. Slowly adapting pulmonary s t r e t c h receptors (SARs) are mechanoreceptors that r e l a t e information concerning the t o t a l volume of gas in the lung ( s t a t i c discharge information) as well as the r a t e a t which the lungs are f i l l e d (dynamic discharge information) ( B a r t l e t t e_t al., 197G). They actually respond to the rate and extent of deformation of pulmonary membranes in which they are s i t u a t e d (Knowlton and Larrabee, 1946; Davis et al.. 1956). C l a s s i c a l l y , slowly adapting pulmonary s t r e t c h receptors were thought to be invoked i n the Hering-Breuer ins p i r a t o r y off-switch reflex. Their input is inhibitory to i n s p i r a t i o n such that once a threshold l e v e l of a f f e r e n t information is attained, i n s p i r a t i o n i s terminated and expiration is promoted (Euler e t al., 1970). Slowly adapting pulmonary s t r e t c h receptors were f i r s t described by Adrian (19 3 3) who recorded from these receptors by way of fi b e r s running in the vagal nerve t r a c t using both cats and rabbits. He i d e n t i f i e d SARs as units that display a regular (tonic) l e v e l of discharge a t end-expiratory lung volume or functional r e s i d u a l capacity (FRO and increase their r a t e of discharge with spontaneous increases in lung volume. It has since been shown that some slowly adapting pulmonary s t r e t c h receptors are tonically active a t functional r e s i d u a l capacity while others only repond when lung volume exceeds FRC (for review see Sant'Ambrogio, 198 2). Studies on mammals have revealed that SARs that show d i f f e r e n t discharge 7 t h r e s h o l d s are anatomically d i s t i n c t r e c e p t o r populations. Slowly adapting pulmonary s t r e t c h r e c e p t o r s t h a t do not e x h i b i t a t o n i c l e v e l of discharge a t FRC tend to be c o n c e n t r a t e d i n the d i s t a l intra-pulmonary airways while SARs t h a t do e x h i b i t a t o n i c l e v e l o f discharge a t FRC are found p r i m a r i l y i n the l a r g e r diameter (usually extrapulmonary) airways ( P a i n t a l , 1977). Since the o r i g i n a l d e s c r i p t i o n o f slowly adapting pulmonary s t r e t c h r e c e p t o r s (Adrian, 1933), i t has been shown t h a t r e c e p t o r discharge corresponds more c l o s e l y with t r a n s m u r a l or transpulmonary p r e s s u r e than with lung volume (Davis e_t a l . . 1956; Sant'Ambrogio e t a l . , 1974; B a r t l e t t e t a l . , 1976; Davenport e t a l . , 198L). In o r d e r f o r a mechanoreceptor to respond to changes i n p r e s s u r e , a net f o r c e must be developed a c r o s s the t i s s u e w a l l i n which the r e c e p t o r s i t s . In animals such as t u r t l e s , the s t r u c t u r e o f the lung and the l o c a t i o n of the pulmonary s t r e t c h r e c e p t o r s i s such t h a t the r e c e p t o r s do not see a change i n transpulmonary p r e s s u r e with lung i n f l a t i o n / d e f l a t i o n c y c l e s (Gans and Hughes, 1967; Jones and Milsom, 1979). In l i g h t of t h i s phenomenon, i t i s not s u r p r i s i n g then to f i n d t h a t t u r t l e SAR discharge p a r a l l e l s changes i n lung volume r a t h e r than ( a r t i f i c i a l l y induced) changes i n transpulmonary p r e s s u r e (Jones and Milsom. 1979). Whether the discharge o f a pulmonary mechanoreceptor corresponds more r e a d i l y to changes i n transpulmonary p r e s s u r e or to changes i n lung volume i s l i k e l y to be dependent on the o r i e n t a t i o n o f the r e c e p t o r within the pulmonary t i s s u e . 8 Receptors t h a t respond to changes i n transpulmonary p r e s s u r e appear to be s i t u a t e d p a r a l l e l to pulmonary muscle f i b e r s and are t h e r e f o r e a c t i v a t e d when they are s t r e t c h e d during muscle e x t e n s i o n ( B a r t l e t t e_ a l . , 1976). Receptors whose discharge corresponds more r e a d i l y to changes i n lung volume are l i k e l y to be l o c a t e d i n s e r i e s with pulmonary muscle f i b e r s and are a c t i v a t e d by t i s s u e compression with changes i n lung volume. I n t e r a c t i o n s Between the Pneumotaxic Center and SARs St i m u l a t i o n o f the nucleus p a r a b r a c h i a l i s medialis, l o c a t e d i n the mid-pontine r e g i o n of the brainstem, r e s u l t s i n the premature t e r m i n a t i o n o f the i n s p i r a t o r y phase o f the br e a t h i n g c y c l e . A more pronounced stimulus i s r e q u i r e d to terminate i n s p i r a t i o n i n the e a r l y phase of the breathing c y c l e than i s r e q u i r e d i n the l a t e r p o r t i o n (Euler e t a l . , 1976). This r e g i o n o f the brainstem, commonly r e f e r r e d to as the pneumotaxic c e n t e r , plays a major r o l e i n v e n t i l a t o r y timing ( M i t c h e l l , 1980). Neurons within the nucleus p a r a b r a c h i a l i s medialis only assume an independent r e s p i r a t o r y rhythm i n the absence o f v a g a l input. During normal r e s p i r a t i o n , the pneumotaxic c e n t e r works i n con j u n c t i o n with a f f e r e n t impulses a r i s i n g from slowly adapting pulmonary s t r e t c h r e c e p t o r s to help determine the timing (duration o f the i n s p i r a t o r y and e x p i r a t o r y phases) o f the brea t h i n g c y c l e ( M i t c h e l l , 1980). S e v e r a l models have been proposed to exp l a i n the i n t e r a c t i o n between pulmonary a f f e r e n t i n f o r m a t i o n (from SARs) and the pneumotaxic c e n t e r , and t h e i r subsequent r o l e s i n the pr o d u c t i o n 9 and maintenance of the breathing p a t t e r n (Clark, and E u l e r , 1972; Feldman, 1976; Agostoni e_t a l . , 1985). The fundamental b a s i s f o r the c o n t r o l o f v e n t i l a t o r y timing i s by no means agreed upon. However, there i s no doubt t h a t v a g a l i n f o r m a t i o n a r i s i n g from slowly adapting pulmonary s t r e t c h r e c e p t o r s plays an important r o l e i n the maintenance of a normal breathing p a t t e r n (Bradley, 1977). Influences on SAR Discharge - E f f e c t s o f Hypercapnia J u s t as many f a c t o r s a f f e c t the f i n a l output of the c e n t r a l rhythm ge n e r a t o r , s e v e r a l f a c t o r s a f f e c t the discharge c h a r a c t e r i s t i c s o f pulmonary s t r e t c h r e c e p t o r s . One such f a c t o r i s C02. Slowly adapting pulmonary s t r e t c h r e c e p t o r discharge i s decreased by v a r i a b l e amounts with i n c r e a s e s i n the C02 c o n c e n t r a t i o n o f i n s p i r e d a i r (Mustafa and Purves, 1972; B a r t l e t t and Sant'Ambrogio, 1976; Jones and Milsom, 1979; M i t c h e l l e_t a l . , 1980). In mammals, intrapulmonary (bronchial) slowly adapting pulmonary s t r e t c h r e c e p t o r s show a much more marked decrease i n discharge with a hypercapnic stimulus than do extrapulmonary (tracheaD SARs (for review see Sant'Ambrogio, 1982). Furthermore, b r o n c h i a l SAR dishcarge decreases much more during l o c a l hypercapnic exposure (increased l e v e l s of i n s p i r e d C02) than with systemic hypercapnic exposure ( B a r t l e t t and Sant'Ambrogio, 1979). Green e_t a l . , (1986) have demonstrated t h a t SARs l o c a t e d i n the lungs o f dogs are i n f a c t s e n s i t i v e to i n c r e a s e d l e v e l s o f pulmonary a r t e r i a l PC02. Since they observed no concomitant change i n t r a c h e a l p r e s s u r e , they assumed t h a t C02 10 evoked the decrease i n SAR discharge by a c t i n g d i r e c t l y on the r e c e p t o r i t s e l f . The a c t u a l mechanism by which C02 e x e r t s i t s e f f e c t s on SAR discharge i s unknown. I t has been proposed t h a t C02 may a f f e c t the microenvironment of the e x t r a c e l l u l a r f l u i d surrounding the s t r e t c h r e c e p t o r . Sant'Ambrogio e_ a l . (1974) have p o s t u l a t e d t h a t the decrease i n pH of the microenvironment a r i s i n g from the i n c r e a s e i n the (H+) a s s o c i a t e d with hypercapnia may be i n s t r u m e n t a l i n the r e d u c t i o n o f SAR discharge. Another p o s s i b l e mechanism proposed by B a r t l e t t and Sant'Ambrogio (1976), i s t h a t the i o n i c composition o f the ECF may be a l t e r e d by a hypercapnic induced r e l e a s e o f protein-bound Ca + + from the r e c e p t o r membrane. This change i n ECF composition may be i n s t r u m e n t a l i n diminishing the response of slowly adapting pulmonary s t r e t c h r e c e p t o r s i n the presence of i n c r e a s e d l e v e l s o f i n s p i r e d C02 ( B a r t l e t t and Sant'Ambrogio, 1976). S t i l l another p o s t u l a t e d mechanism i s t h a t CO2 a f f e c t s the tone of the smooth muscle i n which the r e c e p t o r i s s i t u a t e d whereby a f f e c t i n g i t s discharge ( M i t c h e l l §__ a l . . 1980; Richardson e_t a l . , 1984). Influences on SAR Discharge - E f f e c t s o f Adaptation Slowly adapting pulmonary s t r e t c h r e c e p t o r discharge decreases over time with a maintained stimulus o f e l e v a t e d lung volume (VLE) (Adrian, 19 3 3). This p r o c e s s o f s t r e t c h r e c e p t o r a d a p t a t i o n i s a complex phenomenon which has both a mechanical and an i o n i c b a s i s . The mechanical b a s i s o f mechanoreceptor a d a p t a t i o n u s u a l l y i n v o l v e s a change i n the v i s c o - e l a s t i c 11 p r o p e r t i e s o f the t i s s u e i n which the s t r e t c h r e c e p t o r i s l o c a t e d (Davenport e t aL, 1981; Muza and F r a z i e r , 198 3). The i o n i c b a s i s in v o l v e d i n s t r e t c h r e c e p t o r a d a p t a t i o n can a c t e i t h e r a t the l e v e l o f the sensory ending (which would a f f e c t the spike g e n e r a t o r p o t e n t i a l ) or a t the l e v e l o f the spike encoder (which would e l i c i t p o s tspike decreases i n r e c e p t o r e x c i t a b i l i t y ) (Grigg, 1986). Sensory ending s t r e t c h r e c e p t o r a d a p t a t i o n may involve c a t i o n gated mechanisms (possibly Ca++ gated K+ channels) which are a c t i v a t e d by the changes i n membrane p o t e n t i a l a s s o c i a t e d with d e p o l a r i z a t i o n . These c a t i o n gated mechanisms would se r v e to r e p o l a r i z e the r e c e p t o r membrane and t h e r e f o r e reduce the ge n e r a t o r p o t e n t i a l thus leading to adaptation. S t r e t c h r e c e p t o r a d a p t a t i o n a t the spike encoder l e v e l i s thought to be the r e s u l t of an e l e c t r o g e n i c Na+ pump t h a t i s st i m u l a t e d by the Na+ i n f l u x during the d e p o l a r i z i n g phase o f the pre v i o u s a c t i o n p o t e n t i a l (for a review see Grigg, 1986). The r a t e a t which slowly adapting pulmonary s t r e t c h r e c e p t o r s adapt to a maintained lung i n f l a t i o n i s commonly used as a ba s i s f o r SAR c l a s s i f i c a t i o n (Knowlton and Larrabee, 1946; Davis e t a l . , 1956). These e a r l y c a l c u l a t i o n s o f SAR a d a p t a t i o n i n d i c e s were based on v e r y s h o r t periods o f a maintained e l e v a t e d lung volume stimulus (seconds to minutes). More r e c e n t s t u d i e s undertaken to examine the a d a p t a t i o n responses of SARs to longer term maintained lung i n f l a t i o n s have produced some v e r y i n t e r e s t i n g and seemingly c o n t r o v e r s i a l r e s u l t s . 12 J. Breuer p o s t u l a t e d t h a t the t o n i c component of SAR discharge i n f l u e n c e s the c o n t r o l of v e n t i l a t o r y frequency i n the absence of any phasic component ( B a r t o l i e_t aL, 197 3). B a r t o l i e t aL, (197 3) undertook experiments to examine the hypothesis t h a t changing the t o n i c l e v e l o f SAR discharge (by changing the e n d - e x p i r a t o r y lung volume) i n f l u e n c e s the timing o f the b r e a t h i n g c y c l e . In t h e i r study, c e n t r a l r e s p i r a t o r y rhythm ( v e n t i l a t o r y e f f o r t ) was a s s e s s e d by way of a phrenic neurogram while the e f f e c t o f changing FRC on v e n t i a l t o r y timing was examined i n p a r a l y z e d dogs on cardiopulmonary bypass, i n the presence and absence of v a g a l information. Abolishing the phasic component of PSR discharge by neuromuscular blockade (elimination o f b reathing movements) prolonged the i n s p i r a t o r y phase (TI) of the b r e a t h i n g c y c l e . There was no c o n s i s t a n t e f f e c t on the e x p i r a t o r y phase (TE) and the o v e r a l l decrease i n v e n t i l a t o r y frequency with ensuing p a r a l y s i s was a t t r i b u t e d to an i n c r e a s e i n TI. A l t e r a t i o n s o f f u n c t i o n a l r e s i d u a l c a p a c i t y (maintained f o r 40 to 120 seconds) i n the absence of breathing movements had no a f f e c t on TI but did change o v e r a l l v e n t i l a t o r y frequency by e x e r t i n g a profound e f f e c t on TE. In both p r o t o c o l s , b i l a t e r a l vagotomy abolished the e f f e c t s of changes i n FRC. These r e s u l t s s t r o n g l y i n d i c a t e t h a t a t l e a s t i n dogs, phasic i n f o r m a t i o n p r i m a r i l y a f f e c t s TI and t o n i c i n f o r m a t i o n a f f e c t s TE. Furthermore, TI and TE are under d i f f e r e n t c o n t r o l mechanisms. G r u n s t e i n e t a l . , (1975) performed a d d i t o n a l experiments to f u r t h e r a s s e s s the e f f e c t o f p o s i t i v e e n d - e x p i r a t o r y loading on 13 v e n t i l a t o r y timing r e l a t i o n s h i p s while r e c o r d i n g from slowly adapting pulmonary s t r e t c h r e c e p t o r s i n the cat. The r e s u l t s obtained from these s t u d i e s were i n c l o s e agreement to those obtained by B a r t o l i et. a l . , (197 3). Since s t u d i e s performed by B a r t o l i et_ (1973) showed t h a t i n c r e a s i n g FRC had no e f f e c t on TI, G r u n s t e i n §_ _., (1975) p o s t u l a t e d t h a t t h e r e was e i t h e r a p e r i p h e r a l (SAR) and/or a c e n t r a l ( r e s p i r a t o r y rhythm generator) a d a p t a t i o n to the new l e v e l of lung volume. They examined the discharge of slowly adapting pulmonary s t r e t c h r e c e p t o r s i n c a t s t h a t were spontaneously b r e a t h i n g from f u n c t i o n a l r e s i d u a l c a p a c i t y and then from an e l e v a t e d lung volume (VLE). When animals were bre a t h i n g from FRC, o c c l u s i o n of the t r a c h e a a t e n d - e x p i r a t o r y lung volume did not bring about discharge from the s t r e t c h r e c e p t o r s . Occlusion a t end i n s p i r a t i o n however, did e l i c i t discharge from the SARs (there was t o n i c SAR discharge a t the e n d - i n s p i r a t o r y lung volume but not a t FRC). When animals were b r e a t h i n g from an e l e v a t e d lung volume (achieved by p o s i t i v e e n d - e x p i r a t o r y p r e s s u r e ) , s i m i l a r r e s u l t s were obtained. Upon t r a c h e a l o c c l u s i o n a t e n d - e x p i r a t o r y volume, t h e r e was no t o n i c SAR dishcarge, whereas t r a c h e a l o c c l u s i o n a t e n d - i n s p i r a t o r y volume was accompanied by discharge. Lung volumes a t e n d - i n s p i r a t i o n when breathing from FRC were not appreciably d i f f e r e n t from lung volumes a t e n d - e x p i r a t i o n while b r e a t h i n g from VLE. Since slowly adapting pulmonary s t r e t c h r e c e p t o r s demonstrated t o n i c discharge a t e n d - i n s p i r a t i o n but not a t e n d - e x p i r a t i o n a t VLE, the t o n i c component o f SAR discharge 14 must have adapted to the e l e v a t e d lung volume. G r u n s t e i n e_t a l . , (1975) concluded t h a t the t o n i c component o f the v a g a l mechanism c o n t r o l l i n g TI adapted such t h a t only phasic i n f o r m a t i o n was important i n determining v e n t i l a t o r y timing i.e. TI was independent o f f u n c t i o n a l r e s i d u a l c a p a c i t y . The conclusions of G r u n s t e i n e t a l . , (1975) a l s o e x p l a i n why i n the study of B a r t o l i e t a l . , (197 3), changes i n f u n c t i o n a l r e s i d u a l c a p a c i t y had no e f f e c t on TI. The study o f G r u n s t e i n e_t aL, (1975), however, has a l s o produced c o n f l i c t i n g r e s u l t s concerning the r o l e o f t o n i c SAR discharge i n the modulation o f v e n t i l a t o r y timing. One one hand, they have shown t h a t the t o n i c component of SAR discharge adapted to the new (elevated) lung volume. On the o t h e r hand, the d u r a t i o n o f the e x p i r a t o r y phase o f the breathing c y c l e was extended when the animals were bre a t h i n g from t h i s e l e v a t e d FRC. Therefore, TE must be dependent not only upon phasic lung i n f o r m a t i o n (as was p r e v i o u s l y demonstrated by C l a r k and E u l e r , 1972), but on a s e p a r a t e v a g a l mechanism which i s s e n s i t i v e to abso l u t e e n d - e x p i r a t o r y lung volume ( G r u n s t e r i n e t a l . , 1975). In preliminary s t u d i e s examining c e n t r a l p a t t e r n g e n e r a t o r c o n t r i b u t i o n s to br e a t h i n g p a t t e r n changes induced by a l t e r a t i o n s i n FRC, Stanley e_t a l . , (1975) have demonstrated t h a t with extended periods o f maintained i n f l a t i o n (up to 8 minutes), the phrenic a c t i v i t y (monitoring c e n t r a l r e s p i r a t o r y b r e a t h i n g e f f o r t s ) r e t u r n e d toward normal values. Time course a n a l y s i s i n d i c a t e d t h a t t h i s c e n t r a l a d a p t a t i o n was independent o f the 16 a d a p t a t i o n seen i n the slowly adapting pulmonary s t r e t c h r e c e p t o r s . Extending these f i n d i n g s , one might p o s t u l a t e t h a t the changes i n br e a t h i n g p a t t e r n (the TI and TE timing r e l a t i o n s h i p ) observed with maintained VLE i n the experiments o f B a r t o l i e t a l . , (1973) and G r u n s t e i n e_t a l . , (1975) were only t r a n s i e n t , and i n time they would r e v e r t to the normal p a t t e r n . Jammes et_ a l . , (1983) have suggested t h a t p a r t of the c e n t r a l a d a p t a t i o n seen during maintained VLE i s due to the a c t i v a t i o n o f p r o p r i o c e p t i v e a f f e r e n t s i n the e x p i r a t o r y muscles. These a f f e r e n t s p r o j e c t onto s u p r a s p i n a l s t r u c t u r e s l o c a l i z e d to the medullary i n s p i r a t o r y neurons o f the DRG. S u f f i c i e n t s t i m u l a t i o n of these a f f e r e n t s w i l l p r e m a t u r a l l y terminate TI (Shannon, 1980). If these a f f e r e n t f i b e r s are st i m u l a t e d during the e x p i r a t o r y phase, TE w i l l a l s o be extended (Remmers and M a r t i l l a , 1975). I n t e r c o s t a l p r o p r i o c e p t i v e a f f e r e n t f i b e r s have been shown to be a c t i v a t e d by i n c r e a s e s i n f u n c t i o n a l r e s i d u a l c a p a c i t y (through the process o f e n d - e x p i r a t o r y loading) and cannot be ignored as a p o s s i b l e c o n t r i b u t o r to c e n t r a l r e s p i r a t o r y rhythm h a b i t u t a t i o n . While the s t u d i e s t h a t demonstrate an a d a p t a t i o n or r e s e t t i n g o f the c e n t r a l r e s p i r a t o r y rhythm g e n e r a t o r (Stanely e_t a l . , 1975; Jammes e t aL, 198 3) remain n e a r l y uncontested, a c o n t r o v e r s l y s t i l l e x i s t s as to the degree o f a d a p t a t i o n demonstrated by the slowly adapting pulmonary s t r e t c h r e c e p t o r s with maintained VLE. G r u n s t e i n e t a l . , (1975) concluded t h a t the t o n i c component o f SAR discharge completely adapts to a change i n 16 lung volume and these r e c e p t o r s e s s e n t i a l l y r e s e t t h e i r t h r e s h o l d l e v e l of discharge. These r e s u l t s are i n c o n t r a s t to o t h e r s t u d i e s which show t h a t SARs only p a r t i a l l y adapt to maintained e l e v a t e d lung volumes. D'Angelo and Agostoni (1975) undertook s t u d i e s to determine the e f f e c t s o f changing f u n c t i o n a l r e s i d u a l c a p a c i t y on lung volume - v e n t i l a t o r y timing r e l a t i o n s h i p s . They wanted to a s s e s s the c o n t r i b u t i o n o f slowly adapting pulmonary s t r e t c h r e c e p t o r s to the breathing p a t t e r n changes a s s o c i a t e d with these a l t e r a t i o n s i n FRC. Experiments were conducted on a n e s t h e t i z e d , spontaneously br e a t h i n g c a t s , dogs and r a b b i t s . F u n c t i o n a l r e s i d u a l lung volume was a l t e r e d by having the animals breathe a t e i t h e r continuous p o s i t i v e or negative p r e s s u r e . Clark and E u l e r (1972) have p r e v i o u s l y demonstrated t h a t the normal t i d a l volume - i n s p i r a t o r y d u r a t i o n (VT-TI) r e l a t i o n s h i p i s a hyperbic fun c t i o n . D'Angelo and Agostoni (1975) showed t h a t changing the f u n c t i o n a l r e s i d u a l lung volume of these animals changed the slope o f t h i s h y p e r b o l i c f u n c t i o n and r e s u l t e d i n a l t e r a t i o n s i n the b r e a t h i n g p a t t e r n . When FRC was i n c r e a s e d , t h e r e was an i n c r e a s e i n t i d a l volume and a decrease i n b r e a t h i n g frequency (primarily produced by an e x t e n s i o n of the e x p i r a t o r y phase of the b r e a t h i n g cycle). These p a t t e r n changes r e s u l t e d i n an i n c r e a s e i n the slope of the VT-TI r e l a t i o n s h i p . B i l a t e r a l c e r v i c a l vagotomy eliminated these responses to changes i n FRC i n d i c a t i n g t h a t v a g a l i n f o r m a t i o n a r i s i n g from slowly adapting pulmonary s t r e t c h r e c e p t o r s was r e s p o n s i b l e f o r these changes i n 17 breathing pattern. Furthermore, because these breathing pattern changes were p e r s i s t a n t (lasting a t l e a s t 10 minutes) the SARs could not be adapting to the imposed changes in FRC. If the SARs were adapting (as was suggested by Grunstein §_t al., 1975), then the c e n t r a l r e s p i r a t o r y rhythm generator must be i n f l u e n t i a l in maintaining these breathing p a t t e r n changes. Davenport e_t al., (1981) investigated the adaptation responses of slowly adapting pulmonary s t r e t c h receptors located in the extra thoracic trachea of dogs. Upon stimulation (by elongation of the trachealis muscle), these receptors dramatically increased their discharge rate. When muscle s t r e t c h was maintained, the receptors showed a rapid adaptation response so that a f t e r 3 minutes of muscle s t r e t c h the rate of discharge had adapted to within 50% of the p r e - s t r e t c h values. Davenport _ t al., (1981) a t t r i b u t e d the majority of this adaptation response to changes in the mechanical properties of the muscle in which the receptors were situated. Muza and Frazier (198 3) f u r t h er investigated SAR adaptation by examining the response of these receptors to prolonged s h i f t s in functional r e s i d u a l capacity in spontaneously breathing cats. They recorded from single unit SARs and monitored the discharge of these receptors while the animals underwent spontaneous breathing episodes a t r e s t i n g and then a t a r t i f i c i a l l y elevated lung volumes. Results from this study are consistant with those from Davenport e_t al., (1981) in that they indicate that the receptors underwent only p a r t i a l (albiet significant) adaptation. 18 The c o n f l i c t i n g r e s u l t s regarding the adaptive p r o p e r t i e s of slowly adapting pulmonary s t r e t c h r e c e p t o r s have led to d i f f e r e n t conclusions on the r e l a t i v e importance of the t o n i c (volume r e l a t e d ) and the phasic (rate r e l a t e d ) components of SAR discharge i n the c o n t r o l of the b r e a t h i n g p a t t e r n , p a r t i c u l a r l y a t lung volumes ot h e r than f u n c t i o n a l r e s i d u a l c a p a c i t y . Studies which have shown slowly adapting pulmonary s t r e t c h r e c e p t o r s to adapt completely to maintained changes i n lung volume would tend to suggest t h a t the important component of SAR discharge i s the phasic component (that which monitors changes i n lung volume). This c o n c l u s i o n , i n combination with r e s u l t s demonstrating t h a t the c e n t r a l r e s p i r a t o r y rhythm g e n e r a t o r o f t e n adapts to changes i n lung volume leads one to the c o n c l u s i o n t h a t t o t a l lung volume a c t u a l l y has v e r y l i t t l e importance i n the c o n t r o l o f v e n t i l a t i o n under s t e a d y - s t a t e conditions. In l i g h t o f the o t h e r s t u d i e s t h a t show only a p a r t i a l a d a p t a t i o n o f slowly adapting pulmonary s t r e t c h r e c e p t o r s to maintained VLE, the r o l e of the t o n i c component of SAR discharge i s f a r from defined. In mammals, adjustments i n p o s t u r e or body p o s i t i o n o f t e n produce minor d e v i a t i o n s i n r e s t i n g lung volume (Grunstein e_ a l . , 1975). However, i t i s u n l i k e l y t h a t these animals experience v e r y severe changes i n FRC under normal p h y s i o l o g i c a l conditions. Animals such as t u r t l e s on the o t h e r hand a c t i v e l y undergo s i g n i f i c a n t changes i n t h e i r r e s t i n g lung volumes. T u r t l e s are a q u a t i c animals t h a t spend a g r e a t d e a l o f time p a s s i v e l y f l o a t i n g a t the s u r f a c e of the water. They are a l s o capable o f 19 undergoing v e r y long dives and remain a t v a r i o u s depths i n the water column f o r extended periods of time. These animals are able to a t t a i n these v a r i o u s buoyancy s t a t e s by a l t e r i n g t h e i r s p e c i f i c g r a v i t y through adjustments i n lung volume. Regulation o f lung volume i s brought about by changes i n pulmonary smooth muscle tonus (Milsom, 1975). The normal breathing p a t t e r n o f t u r t l e s c o n s i s t s o f a s e r i e s o f breaths s e p a r a t e d by a v a r i a b l e n o n - v e n t i l a t o r y (breathhold) p e r i o d beginning a t the end o f i n s p i r a t i o n . The o v e r a l l frequency component of t h i s p e r i o d i c b r e a t h i n g p a t t e r n i s a combination o f the number of b r e a t h s within the v e n t i l a t o r y episode and the number of b r e a t h i n g episodes per u n i t time. S e v e r a l s t u d i e s have now shown t h a t the d u r a t i o n o f the n o n v e n t i l a t o r y p e r i o d (or the number of b r e a t h i n g episodes per u n i t time) i s the major c o n t r o l l e d v a r i a b l e determining breathing frequency (Glass e_ a l . , 1978; B e n c h e t r i t and Dejours, 1980; Milsom and Jones, 1980). C o n t r o l o f the d u r a t i o n of the n o n v e n t i l a t o r y p e r i o d i n animals e x h i b i t i n g a p e r i o d i c breathing p a t t e r n i s a problem t h a t has r e c e i v e d a good d e a l of a t t e n t i o n . Since blood gas tensions must change during the breathhold p e r i o d , p e r i p h e r a l and c e n t r a l chemoreceptor r e f l e x i n f o r m a t i o n may be an important f a c t o r i n determining the breathhold length (Burggren e t a l . , 1978; Ackerman and White, 1979; Glass e t a l . , 1983). However, no c l e a r blood gas t e n s i o n t h r e s h o l d s have been defined. Johansen (1970) has suggested t h a t the p r o g r e s s i v e decrease i n lung volume over the d u r a t i o n o f the breathhold p e r i o d (due to 20 cutaneous C02 elimination and blood and tissue C02 storage) may be instrumental i n terminating the breathhold. However, c o r r e l a t i o n studies have revealed that there is a high degree of v a r i a b i l i t y between r e s t i n g lung volumes and breathhold duration (Milsom and Johansen, 1975; Burggren and Shelton, 1979; Ackerman and White, 1979). It i s likely that breathhold duration is controlled through a combination of chemoreceptor information and a f f e r e n t information arising from slowly adapting pulmonary s t r e t c h receptors (Milsom and Chan, 1986). Milsom and Chan (1986) examined the e f f e c t s of chronic changes in r e s t i n g lung volume (VLR) on the v e n t i l a t o r y p a t t e r n of t u r t l e s . When the body weight of these animals was a l t e r e d through the attachment of either weights or f l o a t s to their shells, t u r t l e s increased or decreased their r e s t i n g lung volume. They underwent this physiological adaptation in order to maintain n e u t r a l buoyancy which was normally attained within 24 to 48 hours (Milsom and Chan, 1986). Results from this study indicate that chronic a l t e r a t i o n s in r e s t i n g lung volume had no e f f e c t on the o v e r a l l levels of minute ventilation, t i d a l volume or breathing frequency. There was however, a d i s t i n c t change in the breathing p a t t e r n of these animals. With an increase in r e s t i n g lung volume, the t u r t l e s showed a decrease in the r e l a t i v e t i d a l volume (calculated as t i d a l volume per ml r e s t i n g lung volume), an increase in the duration of the breathhold period and an increase in the number of breaths within each v e n t i l a t o r y period. Increases in VLR may allow the animal to extend the breathhold 21 p e r i o d (due to an i n c r e a s e i n lung oxygen s t o r e s ) but, i n order to maintain an adequate t u r n o v e r of the r e s p i r a t o r y medium i n l i g h t o f the decrease i n the r e l a t i v e t i d a l volume, the number of b r e a t h s within each br e a t h i n g episode must be increased. Unlike the t r a n s i e n t changes i n the mammalian breathing p a t t e r n a s s o c i a t e d with a l t e r a t i o n s o f f u n c t i o n a l r e s i d u a l c a p a c i t y , the p a t t e r n changes i n t u r t l e s p e r s i s t e d f o r many days (Milsom and Chan, 1986). The r e s u l t s of Milsom and Chan (1986) demonstrate t h a t the breathing p a t t e r n trends a s s o c i a t e d with c h r o n i c a l t e r a t i o n s i n lung volume are l i k e l y to be due to the e f f e c t o f these volume changes on pulmonary s t r e t c h r e c e p t o r output. If i n f a c t slowly adapting pulmonary s t r e t c h r e c e p t o r s play a major r o l e i n producing and maintaining the b r e a t h i n g p a t t e r n changes a s s o c i a t e d with long term lung volume a l t e r a t i o n s i n these animals, one would p o s t u l a t e t h a t these r e c e p t o r s must not adapt completely during maintained lung i n f l a t i o n . Thus the p r e s e n t study was undertaken to determine the e f f e c t s of maintained lung i n f l a t i o n on the discharge of slowly adapting pulmonary s t r e t c h r e c e p t o r s . Since i n c r e a s e d l e v e l s o f i n s p i r e d C02 are known to diminish SAR discharge (Jones and Milsom, 1979), the e f f e c t s o f prolonged i n f l a t i o n with hypercapnic gas (5% C02 i n a i r ) on the discharge of these r e c e p t o r s was a l s o i n v e s t i g a t e d . 22 MATERIALS & METHODS Animals: Experiments were performed on freshwater t u r t l e s (Pseudemvs  scr ipta and Chrysemys picta) ranging in weight from 500 to 1000 grams. The t u r t l e s were housed a t room temperature i n plexi-glass tanks f i l l e d to a depth of approximately 40 cm with fre s h running water. Wooden f l o a t s and heat lamps maintained on a 12 hour light cycle were provided for basking. Throughout the winter months the t u r t l e s r a r e l y ate while during the summer months they were o f f e r e d a diet consisting of raw meat (ground beef, l i v e r and brine shrimp). Surgical Procedure: Turtles were single-pithed and restrained. A mid-line incision was made on the v e n t r a l surface of the neck and the trachea was exposed. A cannula was in s e r t e d into the trachea as low in the neck as possible and tie d i n place with s u r g i c a l silk. The incision was then sutured shut and the animals were a r t i f i c i a l l y v e n t i l a t e d with a i r using a Harvard Respirator. To connect a pressure transducer (Statham P23V) to the t r a c h e a l cannula, a small hole was d r i l l e d into the closed end of a p l a s t i c 3-way stopcock and a piece of PE90 tubing was in s e r t e d into this opening and secured with epoxy glue. The tube was f i l l e d with water and connected to the pressure transducer for measurements of i n t r a t r a c h e a l pressure. The pressure signal was simultaneously recorded on magnetic tape (Hewlett-Packard 3968A 23 Instrumentation Recorder) and on a chart recorder (Beckman). The animals were then positioned in dorsal recumbancy to prepare for nerve recordings. Measurement of Pulmonary Stretch Receptor Activity: An incision was made in either the right or left side of the neck beginning just below the ear and extending down the neck for approximately 4 cm. The vagosympathetic nerve (tenth cranial nerve) was kept intact and dissected free from the surrounding tissue. The nerve was laid across a metal base plate and covered with mineral oil. The nerve was transected and then desheathed using fine watch makers forceps. A small cut was made in the nerve from which fiber slips were teased away from the vagal trunk. Single or pauci unit recordings arising from receptors modulated by lung inflation were made using bipolar silver electrodes. The electrical activity from these receptors was amplified, filtered (FRAMP general purpose amplifier; F. Smith, Vancouver, B.C.) and recorded on magnetic tape. The filtered signal was passed through a window discriminator (W.P. Instruments - Model 121) and the output from the window discriminator was counted with a rate meter (EKE Electronics -Model RT 682). The outputs from both the window discriminator and the rate meter were also recorded on the chart recorder while only the signal from the window discriminator was stored on magnetic tape. Throughout the experiments, both the filtered and the unfiltered electrical signals arising from the receptors, the w i n d o w d i s c r i m i n a t o r o u t p u t a n d t h e p r e s s u r e s i g n a l w e r e v i e w e d o n a s t o r a g e o s c i l l i s c o p e ( T e t r o n i x 5111A) a n d e i t h e r t h e f i l t e r e d o r t h e w i n d o w d i s c r i m i n a t e d s i g n a l s w e r e p l a y e d t h r o u g h a n a u d i o a m p l i f i e r ( G r a s s AM8). T h e r a t e m e t e r w a s c a l i b r a t e d w i t h a s t i m u l a t o r ( G r a s s - M o d e l S 6 C ) p r i o r t o t h e r e c o r d i n g p r o c e d u r e a n d t h e p r e s s u r e t r a n s d u c e r w a s c a l i b r a t e d w i t h a w a t e r m a n o m e t e r ( C F . P a l m e r , L o n d o n ) . M e a s u r e m e n t o f B l o o d a n d L u n g G a s e s : L u n g g a s w a s s a m p l e d f r o m t h e t r a c h e a l c a n n u l a u s i n g a 5 0 m l g l a s s s y r i n g e e q u i p p e d w i t h a 3 - w a y s t o p c o c k . W i t h t h e p u m p ventilator o f f and t h e lungs a t resting v o l u m e , g a s w a s d r a w n o u t o f t h e l u n g s b y m a n i p u l a t i n g t h e s y r i n g e a n d t h e s t o p c o c k s o t h a t o n l y t h e e n d - t i d a l g a s w a s s a v e d f o r m e a s u r e m e n t s . T h e f r a c t i o n a l c o n c e n t r a t i o n o f o x y g e n a n d c a r b o n d i o x i d e i n t h e l u n g g a s w a s d e t e r m i n e d u s i n g a n 0 2 a n a l y z e r ( B e c k m a n 0 M - 1 D a n d a C 0 2 a n a l y z e r ( B e c k m a n L B - 2 ) . F o r b l o o d s a m p l i n g , a s m a l l h o l e w a s d r i l l e d i n t h e u p p e r m i d d l e p o r t i o n o f t h e c a r a p a c e a n d t h e l e f t s u b c l a v i a n a r t e r y w a s c a n n u l a t e d . T h e c a n n u l a w a s d r a w n o u t o f t h e a n i m a l t h r o u g h a s m a l l h o l e m a d e j u s t b e l o w t h e f r o n t l e f t l e g a n d t h e h o l e i n t h e c a r a p a c e w a s t h e n r e s e a l e d w i t h d e n t a l a c r y l i c . T h e o x y g e n a n d c a r b o n d i o x i d e e l e c t r o d e s w e r e c a l i b r a t e d u s i n g g a s e s o f v a r y i n g C 0 2 c o n c e n t r a t i o n s m i x e d w i t h a p r e c i s i o n g a s m i x i n g p u m p ( R a d i o m e t e r , GMA 2). T h e pH e l e c t r o d e w a s c a l i b r a t e d u s i n g s t a n d a r d i z e d b u f f e r i n g s o l u t i o n s ( R a d i o m e t e r ; S 1 5 0 0 a n d S1510). A l l c a l i b r a t i o n s a n d m e a s u r e m e n t s w e r e d o n e a t r o o m t e m p e r a t u r e ( 2 2 ° C ) . 25 E x p e r i m e n t a l P r o t o c o l : The e x p e r i m e n t a l p r o c e d u r e c o n s i s t e d o f t h r e e s e p a r a t e p r o t o c o l s as i l l u s t r a t e d i n f i g u r e 1. The s h o r t term (150 seconds) m a i n t a i n e d l u n g i n f l a t i o n s and the dynamic l u n g i n f l a t i o n s o c c u r r e d a t the same p o i n t i n each p r o t o c o l . The d i f f e r e n c e between the p r o t o c o l s r e s i d e d i n the one hour p e r i o d o f m a i n t a i n e d l u n g volume. D u r i n g the c o n t r o l p r o t o c o l the l u n g s were m a i n t a i n e d a t r e s t i n g volume f o r one hour, d u r i n g the t e s t p r o t o c o l the l u n g s were m a i n t a i n e d a t an e l e v a t e d volume f o r one hour and d u r i n g the C02 t e s t p r o t o c o l the l u n g s were m a i n t a i n e d a t an e l e v a t e d volume f o r one hour a f t e r the a n i m a l had been v e n t i l a t e d w i t h 5% C02 i n a i r . Between each p r o t o c o l ( c o n t r o l , t e s t and C02 t e s t ) , the a n i m a l s were pump v e n t i l a t e d w i t h a i r f o r a 45 minute r e c o v e r y p e r i o d . R e s t i n g l u n g volumes i n P_ s c r i o t a and C_ p i c t a have been found to be a p p r o x i m a t e l y 12 ml/100 grams ( J a c k s o n , 1971; Milsom, 1975). R e s t i n g l u n g volume i s d e f i n e d as the e q u i l i b r i u m volume a t which the o p p o s i n g f o r c e s o f the t h o r a x and the l u n g s a r e b a l a n c e d ( J a c k s o n , 1971). I n t u r t l e s t h i s volume i s reached when the a n i m a l s a re l y i n g on t h e i r v e n t r a l s u r f a c e w i t h the g l o t t i s open. R e s t i n g l u n g volume r e s u l t s i n an i n t r a t r a c h e a l p r e s s u r e o f 0 cmH20. For the purpose o f t h i s s t u d y , e l e v a t e d l u n g volume has been d e f i n e d as the volume a s s o c i a t e d w i t h an i n t r a t r a c h e a l p r e s s u r e o f 10 cmH20. The e l e v a t e d l u n g volume was m a i n t a i n e d by a c o n t i n u o u s p o s i t i v e p r e s s u r e o f 10 cmH20. An i m a l s were v e n t i l a t e d w i t h 10 b r e a t h s a t a s t a n d a r d i z e d t i d a l volume FIGURE 1 O u t l i n e of Nerve Recording P r o t o c o l Panel A S t a t i c and dynamic i n f l a t i o n s p r i o r to long term maintained lung volume Panel B Long term maintained lung volume at r e s t i n g ( c o n t r o l p r o t o c o l ) and e l e v a t e d ( t e s t p r o t o c o l ) l e v e l s Panel C S t a t i c and dynamic i n f l a t i o n s f o l l o w i n g long term maintained lung volume The C02-test p r o t o c o l c o n s i s t e d of s t a t i c and dynamic i n f l a t i o n s on a i r (panel A) f o l l o w e d by 5 minutes of pump v e n t i l a t i o n s on 5% C02 i n a i r . A f t e r t h i s e q u i l i b r a t i o n p e r i o d , the t e s t p r o t o c o l was performed ( a l l i n f l a t i o n s were with 5% C02 i n a i r ) . CONTROL PROTOCOL B 1 5 _ _ _ v e n t . ! 0 s 1 2 150s vent. 60 minutes at V | _ R E S T II . 30s vent. _ 150s. . 30s. v e n t — 1 2 ON T E S T P R O T O C O L B 1 5^!_ vent. I 0 s -1 2 150s. ,30s. _____ vent.__ ll 1 2 60 minutes at V _ELEVATED 150s. v e n t . i 0 s 1 2 150s. _ 30s v e n t — 27 (2.5 ml/100 grams) which was superimposed on both r e s t i n g and e l e v a t e d lung volumes. When a single u n i t slowly adapting pulmonary s t r e t c h r e c e p t o r was i d e n t i f i e d , the p r o t o c o l was begun. Slowly adapting pulmonary s t r e t c h r e c e p t o r s are e a s i l y recognized as r e c e p t o r s t h a t respond not only to an i n c r e a s e i n lung volume with an i n c r e a s e i n discharge r a t e , but a l s o show a slow r a t e o f a d a p t a t i o n to a maintained e l e v a t i o n i n lung volume (Adrian, 1933; Knowlton and Larrabee, 1946; Davis e_t a l . , 1956). In a d d i t i o n , slowly adapting pulmonary s t r e t c h r e c e p t o r s demonstrate a discharge o v e r s h o o t upon a sudden i n c r e a s e i n lung volume as w e l l as an o f f - r e s p o n s e where discharge i s absent f o r a s h o r t p e r i o d o f time upon lung d e f l a t i o n (Sant'Ambrogio, 1982). I d e a l l y , a l l 3 components of the p r o t o c o l were performed on each single u n i t r e c e p t o r . To complete t h i s t a s k r e c o r d i n g s had to be made conti n u o u s l y f o r a t l e a s t 6 hours. Since t h i s was o f t e n impossible, much of the data i s comprised o f rec o r d i n g s from r e c e p t o r s during only 1 or 2 components of the p r o t o c o l . Since the nerve r e c o r d i n g apparatus i s extremely s e n s i t i v e to movement, the blood and lung gases were analyzed during a se p a r a t e s e r i e s of experiments on a d i f f e r e n t group of t u r t l e s to avoid the r i s k of damaging the r e c o r d i n g p r e p a r a t i o n . Blood samples were withdrawn before beginning the p r o t o c o l as w e l l as p r i o r to and immediately following the e q u i l i b r a t i o n p e r i o d (see f i g u r e 2). Again, animals were v e n t i l a t e d with e i t h e r a i r or 5% C02 i n a i r . The blood and lung gas values obtained during 28 e q u i l i b r a t i o n a t r e s t i n g lung volumes were not s i g n i f i c a n t l y d i f f e r e n t from those obtained during e q u i l i b r a t i o n a t e l e v a t e d lung volumes and only the data from the e l e v a t e d lung volume experiments are presented. Subsequent s t u d i e s were performed using the b a s i c p r o t o c o l with a 6 hour e q u i l i b r a t i o n period. In these experiments, blood samples were removed as d e s c r i b e d above with a d d i t i o n a l samples taken every 2 hours throughout the extended e q u i l i b r a t i o n period. Data A n a l y s i s : Since the data obtained from the two subspecies o f a q u a t i c t u r t l e s used i n t h i s study were not s i g n i f i c a n t l y d i f f e r e n t , the r e s u l t s were combined. Experiments were conducted i n both summer ( A p r i l to September) and winter (October to March) months. Even though the metabolism of these animals tends to be lower during the winter months (Bennett and Dawson, 1976), there were no s e a s o n a l d i f f e r e n c e s i n the data obtained, probably owing to the extreme s t a t e o f c e r e b r a l d i s a r r a y of the animals. To analyze the data, f i l t e r e d e l e c t r i c a l s i g n a l s a r i s i n g from slowly adapting pulmonary s t r e t c h r e c e p t o r s t h a t were p r e v i o u s l y s t o r e d on magnetic tape were played back. These s i g n a l s were r e d i s c r i m i n a t e d and then counted using the r a t e meter. The si g n a l s from the window d i s c r i m i n a t o r , the r a t e meter and the p r e v i o u s l y r e c o r d e d s i g n a l s from the p r e s s u r e t r a n s d u c e r were then t r a n s f e r r e r d to a Gould c h a r t r e c o r d e r . A n a l y s i s of the s t a t i c discharge component i n v o l v e d examining the r a t e of occurance of a c t i o n p o t e n t i a l s i n response to 29 FIGURE 2. Outl ine of Blood Gas and Lung Gas P r o t o c o l Test P r o t o c o l : Panels A, B and C correspond to f i g u r e 1 c a p t i o n s C02-test P r o t o c o l : Panel A S t a t i c and dynamic i n f l a t i o n s on a i r Panel B Long term maintained i n f l a t i o n a f t e r a t l e a s t 5 Panel C S t a t i c and dynamic i n f l a t i o n s f o l l o w i n g long term maintained e l e v a t e d lung volume minutes of pump v e n t i l a t i o n on 5% C02 i n a i r Symbols: denotes lung gas sampling denotes blood gas sampling • 0 • 0 A • 4v-TEST PROTOCOL B 0 •V— I I V— 1 2 A •V-•V- I I T c o 2 - TEST < v— CO2 ADDITION ( 5 min ) B I I • 0 v— maintained i n c r e a s e s and decreases i n lung volume. Discharge r a t e was analyzed a t the following p o i n t s i n the p r o t o c o l : a. r e s t i n g lung volume p r i o r to s t a t i c lung i n f l a t i o n b. peak, discharge with s t a t i c lung i n f l a t i o n c. f o r 2 1/2 minutes a t e l e v a t e d lung volume following the i n i t i a l lung i n f l a t i o n (2,4,6,8,10,20,30,60,90 and 150 seconds) d. f o r 30 seconds following pump v e n t i l a t i o n a t e l e v a t e d lung volume (2,4,6,8,10,20, and 30 seconds) e. f o r 2 1/2 minutes a t r e s t i n g lung volume following lung d e f l a t i o n (as i n c) f. f o r 30 seconds following pump v e n t i l a t i o n a t r e s t i n g lung volume (as i n d) g. p r i o r to the e q u i l i b r a t i o n p e r i o d h. a t 5 minute i n t e r v a l s throughout the e q u i l i b r a t i o n period. Figure 3 i l l u s t r a t e s the poi n t s a t which s t a t i c r e c e p t o r discharge was analyzed i n each component of the p r o t o c o l . A n a l y s i s o f the dynamic discharge a s s o c i a t e d with pump v e n t i l a t i o n i n v o l v e d examining the r a t e of occurance o f a c t i o n p o t e n t i a l s before the v e n t i l a t o r y c y c l e s (baseline discharge), a t peak i n s p i r a t o r y lung volume and en d - e x p i r a t o r y lung volume. The absolute number o f a c t i o n p o t e n t i a l s f i r e d during the i n s p i r a t o r y and e x p i r a t o r y p o r t i o n s o f each v e n t i l a t o r y c y c l e were a l s o counted. 31 Two-way a n a l y s i s of variance was used to t e s t the s t a t i s t i c a l s i g n i f i c a n c e o f the data i n t h i s study. D i f f e r e n c e s were considered s i g n i f i c a n t a t the P<0.05 l e v e l . RESULTS SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS i) Response to Maintained Lung I n f l a t i o n and Deflation Figure 3a i l l u s t r a t e s the discharge c h a r a c t e r i s t i c s of a slowly adapting pulmonary stretch receptor i n response to maintained lung i n f l a t i o n . At FRC (0 cmH20) the receptor exhibited a tonic discharge rate of 1 to 2 Hz. Upon s t a t i c lung i n f l a t i o n to 10 cmH20, the peak i n f l a t i o n discharge rate reached 10 Hz and then slowly declined so that the receptor was f i r i n g at a rate of 6 Hz af t e r 150 seconds of maintained i n f l a t i o n period. Figure 3b demonstrates the response of the same SAR to lung d e f l a t i o n . At maintained lung i n f l a t i o n , the SAR was discharging at a rate of 9 Hz. Upon d e f l a t i o n (to functional residual lung volume) the receptor discharge was abolished for 10 seconds, after which i t returned to the previously recorded rate of 1 to 2 Hz. This off-response was c h a r a c t e r i s t i c of most of the slowly adapting pulmonary stretch receptors recorded from i n this study. i i ) Response to Dynamic Lung I n f l a t i o n and Deflation Figure 4 i l l u s t r a t e s the response of a slowly adapting pulmonary stretch receptor to 10 pump v e n t i l a t i o n cycles superimposed upon resting (airway pressure = 0 cmH20) and then elevated (airway pressure = 10 cmH20) lung volume. Prior to dynamic i n f l a t i o n at resting lung volume, the receptor exhibited a tonic discharge rate of 2 Hz. With increases i n lung volume during the i n f l a t i o n cycle, receptor discharge increased markedly 33 FIGURE 3. Response of SAR #11 to S t a t i c Lung I n f l a t i o n and  D e f l a t i o n 3a maintained i n f l a t i o n a t e l e v a t e d lung volume f o r 150 seconds 3b d e f l a t i o n from e l e v a t e d lung volume (10 cmH20) to r e s t i n g lung volume (0 cmH20) 34 FIGURE 4. Response of SAR #11 to Dynamic Lung I n f l a t i o n s 4a pump v e n t i l a t i o n a t r e s t i n g lung volume 4b pump v e n t i l a t i o n a t e l e v a t e d lung volume PAw(cmH20) Rate (Hz) Window Neurogram o o o o Discriminator i i J i i 35 (mean peak inspiratory discharge rate = 6.9 Hz +/- 0.32 S.D.M.). During lung d e f l a t i o n , the receptor exhibited no baseline discharge even though the same lung volume e l i c i t e d a tonic discharge rate of 2 Hz prior to lung i n f l a t i o n . At elevated lung volume the receptor f i r e d at a tonic discharge rate of 6 Hz. The mean peak inspiratory discharge rate was 14.0 Hz (+/- 0.50 S.D.M.) and the mean expiratory discharge rate was 5.0 Hz (+/- 0.67 S.D.M.), somewhat less than the tonic rate at the same lung volume. Thus the off-response demonstrated in figure 3b also occurred during the dynamic i n f l a t i o n cycles. Two populations of slowly adapting pulmonary stretch receptors were i d e n t i f i e d which we have designated low threshold SARs (n=13) and high threshold SARs (n=4). Low threshold SARs exhibited a tonic rate of discharge at FRC while high threshold SARs only responded when lung volumes exceeded FRC. Aside from volume thresholds, these two populations of receptors had differences i n their adaptation c h a r a c t e r i s t i c s as well as i n their s e n s i t i v i t i e s to increased l e v e l s of inspired C02. LOW THRESHOLD SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS i) Responses to Maintained Lung In f l a t i o n s with Air Figure 5 i l l u s t r a t e s the s t a t i c i n f l a t i o n responses of low threshold slowly adapting pulmonary stretch receptors. Panel A shows the response of these receptors to short term (150 seconds) lung i n f l a t i o n s prior to the long term (1 hour) maintained i n f l a t i o n periods (panel B). Panel C shows the response of these 36 FIGURE 5. Low Threshold F i b e r s ; Response to Maintained S t a t i c  Lung I n f l a t i o n s Panel A Short term i n f l a t i o n (150 seconds) Panel B Long term maintained i n f l a t i o n (60 minutes) Panel C Short term i n f l a t i o n (150 seconds) Discharge i s r e p r e s e n t e d as a per cent of peak di s c h a r g e a t the i n i t i a l p o r t i o n of the p r o t o c o l (panel A) PAW = airway pressure (0 - 10 cmH20) 36a CONTROL PROTOCOL A SiOi r -10 - 0 1CXH UJ 80 CL UJ 60 i o cc % 52CH i — i — i — i — H h — i — i — i R E 10 20 30 SO (0 120 150 Mconds — H h — i — i — i — i — i 25 10 20 30 40 50 SO a i n u U t 100 80 60 -40 -20 I — i — i — i — r l h — i — I — I L 0 R E 10 20 30 60 90 120 1S0 stcandt T E S T P R O T O C O L A s i 1 0 ! ~ n-i —I •10 •o 100 \ I If 80 S 60 i or < x u in 40 -\ 20 H i — i — i — i — r i h — i — i — i R E 10 20 30 00 00 120 BO • • c o n d s i — i — r l h — i — ' — i — r — i R E 2.5 10 20 30 *0 50 SO n i n u t t * I 1 1 1—Hh 1 — i - i R E 10 20 30 SO SO 120 150 M c e n d i 100 80 60 40 20 C02 T E S T PROTOCOL 10 J 100 I 80 a? m 60 5 40 o in ° 20 -10 - 0 (-100 80 NO 40 20 R E • 20 x'so » 120 150 ' * E 2.5"» 20 30 « SO SO k E ,b 90 120 150 0 PRIOR TO C02 AODITION mtnut tc AFTER C02 AOOIT ION I5min.) 37 r e c e p t o r s to s h o r t term i n f l a t i o n s following the 1 hour maintained i n f l a t i o n p e r i o d (see f i g u r e 2 f o r p r o t o c o l outline). In f i g u r e 5 r e c e p t o r discharge i s r e p r e s e n t e d as a per cent of the peak, discharge r a t e . Peak discharge r a t e i s t h a t which was obtained with acute s t a t i c lung i n f l a t i o n a t the beginning of the p r o t o c o l (panel A). In the c o n t r o l p r o t o c o l , peak i n f l a t i o n i s defined as 100% (point E, panel A) and slowly declines over the s h o r t term (150 second) i n f l a t i o n period. At 150 seconds (panel A), the mean discharge r a t e i s 64% of peak. When the lungs were maintained a t r e s t i n g lung volume f o r 1 hour (panel B), discharge remained c l o s e to the baseline discharge l e v e l (point R, panel A). A f t e r the 1 hour e q u i l i b r a t i o n p e r i o d , s t a t i c lung i n f l a t i o n r e s u l t e d i n a mean discharge value t h a t was not s t a t i s t i c a l l y d i f f e r e n t to the same c o n d i t i o n p r i o r to the e q u i l i b r a t i o n p e r i o d (point E, panel C vs panel A). In the t e s t p r o t o c o l , a f t e r 1 hour of maintained i n f l a t i o n on a i r the r e c e p t o r s adapted 80% or to with i n 20% of t h e i r baseline ( f u n c t i o n a l r e s i d u a l lung volume) discharge l e v e l s (60 minutes,panel B). Most of t h i s a d a p t a t i o n o c c u r r e d during the i n i t i a l 2.5 minutes (150 seconds) o f the maintained i n f l a t i o n period. Even with the Large degree o f r e c e p t o r a d a p t a t i o n observed over the 1 hour i n f l a t i o n p e r i o d , d e f l a t i o n to r e s t i n g lung volume (point R, panel C) r e s u l t e d i n e s s e n t i a l l y the same mean t o n i c r a t e o f discharge (panel C). In a d d i t i o n , the mean discharge r a t e s a t peak i n f l a t i o n and a t 150 seconds o f maintained i n f l a t i o n were not s i g n i f i c a n t l y d i f f e r e n t before 38 (panel A) and a f t e r (panel C) long term maintained i n f l a t i o n . However, the r a t e a t which the r e c e p t o r s adapted to the s h o r t term lung i n f l a t i o n stimulus were d i f f e r e n t . Figure 6 shows a d a p t a t i o n i n d i c i e s f o r slowly adapting pulmonary s t r e t c h r e c e p t o r s during s h o r t term lung i n f l a t i o n s p r i o r to and following the long term maintained i n f l a t i o n periods. The a d a p t a t i o n index used i n the analyses o f these data was d e r i v e d from t h a t of Knowlton and Larrabee (1946) and Davis e t a l . , (1956). Mean r e c e p t o r a d a p t a t i o n i n d i c i e s were c a l c u l a t e d f o r 2,4,6,8,10 and 150 seconds following peak i n f l a t i o n (point E) during the s h o r t term i n f l a t i o n s p r i o r to (panel A) and following (panel C) long term maintained lung i n f l a t i o n (panel B). In the t e s t run, SARs demonstrated a more r a p i d r a t e of a d a p t a t i o n following long term lung i n f l a t i o n than p r i o r to t h i s i n f l a t i o n period. The mean a d a p t a t i o n r a t e s to a s h o r t term lung i n f l a t i o n stimulus i n the c o n t r o l p r o t o c o l were e s s e n t i a l l y the same p r i o r to and following a p e r i o d of long term maintainance of r e s t i n g lung volume. ii ) Responses to Maintained Lung I n f l a t i o n s with Increased L e v e l s  o f Inspired C02 A f t e r 1 hour of maintained i n f l a t i o n with hypercapnic a i r (5% C02 i n a i r ) , the slowly adapting pulmonary s t r e t c h r e c e p t o r s adapted 88% or to within 12% o f t h e i r baseline discharge l e v e l s (figure 5, C 0 2 - t e s t p r o t o c o l , panel B). Following the long term maintained lung i n f l a t i o n p e r i o d on 5% C02, d e f l a t i o n to r e s t i n g lung volume l a t e r i n the p r o t o c o l r e s u l t e d i n e s s e n t i a l l y the 39 FIGURE 6 A d a p t a t i o n I n d i c i e s f o r Low Threshold Slowly Adapting  Pulmonary S t r e t c h Receptors A d a p t a t i o n i n d i c i e s f o r s h o r t term i n f l a t i o n s (150 seconds) before and a f t e r the p e r i o d of long term maintained lung volume are s t a t i s t i c a l l y d i f f e r e n t i n the t e s t run a t 6, 8, and 10 seconds. p r i o r to 1 hour maintained lung volume f o l l o w i n g 1 hour maintained lung volume 39a A D A P T A T I O N INDEX = f (PEAK) - f (x sec) x 100 f (PEAK) 60 -40 -20 Control Protocol • a f t e r 1 h o u r m a i n t a i n e d l u n g v o l u m e 8 0 6 0 X= 2 sec . 4 s e c . 6 s e c . 8 s e c . 10sec. 150sec. " T e s t P r o t o c o l < AO 2 0 -X= 2 s e c . A s e c . 6 s e c . 8 sec . 10 s e c 150 sec. 6 ° - CO2 T e s t P r o t o c o l . AO -2 0 2 s e c . L s e c . 6 s e c . 8 s e c . 10 s e c . 150sec. 40 same mean t o n i c r a t e of discharge (point R, panel C). In c o n t r a s t to the t e s t p r o t o c o l , the mean peak i n f l a t i o n discharge r a t e i n panel C o f the C 0 2 - t e s t p r o t o c o l was s i g n i f i c a n t l y l e s s than the mean peak i n f l a t i o n r a t e p r i o r to C02 a d d i t i o n (panel A). The mean values f o r the C 0 2 - t e s t a d a p t a t i o n i n d i c i e s (figure 6) were g r e a t e r following long term maintained i n f l a t i o n on hypercapnic a i r than they were p r i o r to t h i s treatment. i i i ) Responses to Dynamic I n f l a t i o n s with A i r Figure 7 i l l u s t r a t e s the mean baseline (pri o r to pump v e n t i l a t i o n ) , peak i n s p i r a t o r y and end e x p i r a t o r y discharge r a t e s of the slowly adapting pulmonary s t r e t c h r e c e p t o r s . Long term (1 hour) maintenance o f lung volume a t r e s t i n g l e v e l s ( c o n t r o l p r o tocol) did not a f f e c t the mean values f o r base l i n e , peak i n s p i r a t o r y and end e x p i r a t o r y discharge r a t e s o f the SARs a t e i t h e r e l e v a t e d (A vs C) or r e s t i n g (a vs c) lung volumes. Long term maintenance o f lung volume a t e l e v a t e d l e v e l s ( t e s t protocol) did r e s u l t i n s i g n i f i c a n t d i f f e r e n c e s between mean values f o r peak i n s p i r a t o r y and end e x p i r a t o r y discharge r a t e s o f the SARs a t both e l e v a t e d (A vs C) and r e s t i n g (a vs c) lung volumes. There were no d i f f e r e n c e s between mean baseline discharge values a t e i t h e r e l e v a t e d or r e s t i n g lung volumes p r i o r to and fol l o w i n g long term maintained lung i n f l a t i o n . iv) Responses to Dynamic I n f l a t i o n s with Increased L e v e l s of Ins p i r e d CO2 Long term maintenance of lung volume a t e l e v a t e d l e v e l s with hypercapnic a i r (C02-test protocol) did not r e s u l t i n s i g n i f i c a n t 41 FIGURE 7 Low Threshold! F i b e r s : Response _ . Dynamic Luna I n f l a t i o n s ( D i s c h a r g e Rate) Mean d i s c h a r g e r a t e (+/- S.D.M.) f o r pump v e n t i l a t i o n c y c l e s . C o n t r o l p r o t o c o l (n=8 a n i m a l s / 10 pump v e n t i l a t i o n s ) T e s t p r o t o c o l (n=8 a n i m a l s / 10 pump v e n t i l a t i o n s ) C 0 2 - t e s t p r o t o c o l (n=7 a n i m a l s / 10 pump v e n t i l a t i o n s ) A mean d i s c h a r g e a t e l e v a t e d l u n g volume p r i o r t o l o n g term m a i n t a i n e d l u n g volume C mean d i s c h a r g e a t e l e v a t e d l u n g volume f o l l o w i n g l o n g term m a i n t a i n e d l u n g volume a mean d i s c h a r g e a t r e s t i n g l u n g volume p r i o r t o l o n g term m a i n t a i n e d l u n g volume c mean d i s c h a r g e a t r e s t i n g l u n g volume f o l l o w i n g l o n g term m a i n t a i n e d l u n g volume 41 i DYN A MIC INFLATIONS Elevated Lung Volume , Resting Lung Volume 30i 20' c o N 0-5 30' B I E LU O D_ 20. in O 10 CO 0-30' 1 in £ 20 < N o o 10 B I E A B I E • B I E 1 B I E C B i E B B I E B I E [ ± L _ L d B I E 1 B I E B I E B= Baseline I = Peak Inspiration E= Expiration 41-d i f f e r e n c e s between mean discharge values a t e l e v a t e d lung volumes before C02 a d d i t i o n (panel A) or a f t e r maintained i n f l a t i o n with hypercapnic a i r (panel C). However, there was a s i g n i f i c a n t d i f f e r e n c e i n the r e s p e c t i v e baseline values a t r e s t i n g lung volumes (panel a vs panel c). Peak, i n s p i r a t o r y and en d - e x p i r a t o r y values were s i g n i f i c a n t l y d i f f e r e n t a f t e r long term maintained lung i n f l a t i o n on hypercapnic a i r both a t e l e v a t e d (A vs C) and a t r e s t i n g (a vs c) lung volumes, v) Dynamic I n f l a t i o n s - Discharge Rate vs Absolute Number o f  Impulses Figure 8 i l l u s t r a t e s the a b s o l u t e number o f impulses f i r e d during the i n s p i r a t o r y and the e x p i r a t o r y phases of the pump v e n t i l a t i o n c y c l e s . In the c o n t r o l p r o t o c o l , t h e r e was a s i g n i f i c a n t d i f f e r e n c e i n the mean number of impulses f i r e d during the i n s p i r a t o r y phase of the v e n t i l a t o r y c y c l e before and a f t e r the 1 hour p e r i o d o f maintained r e s t i n g lung volume a t both e l e v a t e d (A vs C) and r e s t i n g (a vs c) lung volumes. There was no change i n the mean number o f impulses during the e x p i r a t o r y phase. A f t e r 1 hour o f maintained i n f l a t i o n on a i r ( t e s t p r o t o c o l ) , there was a s i g n i f i c a n t d i f f e r n c e i n the number of impulses f i r e d during both the i n s p i r a t o r y and e x p i r a t o r y phases of the v e n t i l a t o r y c y c l e s a t both e l e v a t e d (A vs C) and r e s t i n g (a vs c) lung volumes. When the lungs were maintained on 5% C02 f o r 1 hour (C02-test p r o t o c o l ) , there was a s i g n i f i c a n t decrease i n the number of impulses f i r e d during i n s p i r a t i o n and e x p i r a t i o n a t e l e v a t e d lung volumes (A vs C) and during i n s p i r a t i o n a t 43 FIGURE 8. Low T h r e s h o l d F i b e r s ; Response t o Dynamic Lung  I n f l a t i o n s ( A b s o l u t e Number o f Impulses) Mean number o f i m p u l s e s (+/- S.E.M.) f o r 10 pump v e n t i l a t i o n s C o n t r o l p r o t o c o l (n=8 a n i m a l s / 10 pump v e n t i l a t i o n s ) T e s t p r o t o c o l (n=8 a n i m a l s / 10 pump v e n t i l a t i o n s ) C 0 2 - t e s t p r o t o c o l (n=7 a n i m a l s / 10 pump v e n t i l a t i o n s ) A mean number o f i m p u l s e s a t e l e v a t e d l u n g volume p r i o r t o l o n g term m a i n t a i n e d i n f l a t i o n C mean number o f i m p u l s e s a t e l e v a t e d l u n g volume f o l l o w i n g l o n g term m a i n t a i n e d i n f l a t i o n a mean number o f i m p u l s e s a t r e s t i n g l u n g volume p r i o r t o l o n g term m a i n t a i n e d i n f l a t i o n c mean number o f i m p u l s e s a t r e s t i n g l u n g volume f o l l o w i n g l o n g term m a i n t a i n e d i n f l a t i o n j a DYNAMIC INFLATIONS Elevated Lung Volume Resting Lung Volume I E I E I E I E I E I E I E I E I E I E I E 1_ I E I = Inspiratory Phase E= Expiratory Phase 44 r e s t i n g lung volumes (a vs c). The decreases i n the mean number of impulses f i r e d during the v e n t i l a t o r y c y c l e i n the C 0 2 - t e s t p r o t o c o l appeared to be g r e a t e r than the decreases seen i n the t e s t p r o t o c o l . HIGH THRESHOLD SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS i) Responses to Maintained Lung I n f l a t i o n s with A i r Figure 9 i l l u s t r a t e s the s t a t i c i n f l a t i o n responses of high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s . These r e c e p t o r s responded to an i n c r e a s e i n lung volume with an in c r e a s e i n discharge r a t e as did the low t h r e s h o l d r e c e p t o r s . However, the t o n i c baseline ( f u n c t i o n a l r e s i d u a l lung volume) discharge l e v e l o f these r e c e p t o r s was always 0 Hz. A f t e r 1 hour of maintained i n f l a t i o n on a i r ( t e s t run), the r e c e p t o r s had adapted to within 75% of t h e i r baseline discharge l e v e l (panel B). Again, as with the low t h r e s h o l d SARs, most o f t h i s a d a p t a t i o n had occured within the f i r s t 2.5 minutes (150 seconds) of the maintained i n f l a t i o n p e r i o d but unlike the low t h r e s h o l d SARs, these r e c e p t o r s did not begin to adapt u n t i l 3 to 4 seconds a f t e r the onset of lung i n f l a t i o n . The mean i n f l a t i o n r a t e s a t peak i n f l a t i o n and a t 150 seconds of maintained i n f l a t i o n did not appear to be d i f f e r e n t before (panel A) and a f t e r (panel C) long term maintained i n f l a t i o n . Furthermore, the r a t e s a t which these r e c e p t o r s adapted to the new l e v e l o f discharge did not appear to be d i f f e r e n t before or a f t e r long term maintained i n f l a t i o n . Because o f the small sample 45 FIGURE 9. High Threshold F i b e r s ; Response to Maintained S t a t i c  Lung I n f l a t i o n s Panel A Short term i n f l a t i o n (150 seconds) Panel B Long term maintained i n f l a t i o n (60 minutes) Panel C Short term i n f l a t i o n (150 seconds) Discharge i s r e s p r e s e n t e d as a per cent of peak d i s c h a r g e a t the i n i t i a l p o r t i o n of the p r o t o c o l (panel A) PAW = airway pressure (0 - 10 cmH20) 46 s i z e i n t h i s p o p u l a t i o n of SARs, a d a p t a t i o n i n d i c e s are not pres e n t e d f o r t h i s r e c e p t o r group. ii ) Responses to Maintained Lung I n f l a t i o n s with Increased L e v e l s of I n s p i r e d C02 A f t e r 1 hour o f maintained i n f l a t i o n with hypercapnic a i r (5% C02 i n a i r ) , the high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s had adapted 55% or to with i n 45% o f t h e i r baseline discharge l e v e l s (figure 9, panel B). The response of the SARs to s t a t i c lung i n f l a t i o n following maintained i n f l a t i o n with 5% C02 i n a i r (panel C, point E) was l e s s than the response to s t a t i c lung i n f l a t i o n p r i o r to C02 a d d i t i o n (panel A, poin t E). The r a t e to which the r e c e p t o r s adapted during the s h o r t term maintained lung i n f l a t i o n was a l s o lower than p r i o r to C02 a d d i t i o n (panel C vs panel A). i i i ) Responses to Dynamic I n f l a t i o n s with A i r Figure 10 i l l u s t r a t e s the mean b a s e l i n e , peak i n s p i r a t o r y and end e x p i r a t o r y discharge r a t e s of the high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s . Long term (1 hour) maintenance o f lung volume a t r e s t i n g l e v e l s ( c o n t r o l p r o t o c o l ) did not appear to a f f e c t the mean values f o r baseline and end e x p i r a t o r y discharge r a t e s o f the SARs a t e i t h e r e l e v a t e d (A vs C) or r e s t i n g (a vs c) lung volumes. The d i f f e r e n c e s i n mean peak i n s p i r a t o r y discharge a t e l e v a t e d lung volumes (A vs C) appear to be due to one p a r t i c u l a r f i b e r . Since the sample s i z e o f t h i s r e c e p t o r p o p u l a t i o n i s small (n=3), t h i s f i b e r has succeeded i n d i s t o r t i n g the mean value. 47 FIGURE 10 High T h r e s h o l d F i b e r s ; Response to Dynamic Lung  I n f l a t i o n s ( D i s c h a r g e Rate) Mean d i s c h a r g e r a t e f o r pump v e n t i l a t i o n c y c l e s . C o n t r o l p r o t o c o l (n=3 a n i m a l s / 10 pump v e n t i l a t i o n s ) T e s t p r o t o c o l (n=2 a n i m a l s / 10 pump v e n t i l a t i o n s ) C 0 2 - t e s t p r o t o c o l (n=2 a n i m a l s / 10 pump v e n t i l a t i o n s ) A mean d i s c h a r g e a t e l e v a t e d l u n g volume p r i o r t o l o n g term m a i n t a i n e d l u n g volume C mean d i s c h a r g e a t e l e v a t e d l u n g volume f o l l o w i n g l o n g term m a i n t a i n e d l u n g volume a mean d i s c h a r g e a t r e s t i n g l u n g volume p r i o r t o l o n g term m a i n t a i n e d l u n g volume c mean d i s c h a r g e a t r e s t i n g l u n g volume f o l l o w i n g l o n g term m a i n t a i n e d l u n g volume 4?a 3 0 1 20-c o ° 1 0 N 0 30-20 LU O cr ts O CO 0 30 n w20 r -O 10 0 DYNAMIC INFLATIONS Elevated Lung Volume A C B I B I E B I E B I C B I E B I Resting Lung Volume a c B I E a B I B I E c B I B B I E B = Baseline I =Peak Inspiration E = Expiration 48 Long term maintenance o f lung volume a t e l e v a t e d l e v e l s ( t e s t p r o t ocol) did not r e s u l t i n any d i f f e r e n c e s between mean values f o r b a s e l i n e , peak, i n s p i r a t o r y or end e x p i r a t o r y discharge r a t e s a t e i t h e r e l e v a t e d or r e s t i n g lung volumes. iv) Responses to Dynamic I n f l a t i o n s with Increased L e v e l s of  Inspi r e d C02 Long term maintenance of lung volume a t e l e v a t e d l e v e l s with hypercapnic a i r (C02-test protocol) did not r e s u l t i n any d i f f e r e n c e between peak i n s p i r a t o r y or end e x p i r a t o r y discharge r a t e s a t e l e v a t e d lung volumes. I t i s i n t e r e s t i n g to note t h a t a t e l e v a t e d lung volumes following long term maintained lung i n f l a t i o n ( C02-test p r o t o c o l , panel C), the mean en d - e x p i r a t o r y discharge r a t e i s lower than the baseline r a t e demonstrating the presence of a dynamic undershoot i n r e c e p t o r discharge. Long term maintenance of lung volume i n the C 0 2 - t e s t p r o t o c o l did not appear to have any e f f e c t on the mean b a s e l i n e , peak i n s p i r a t o r y or end e x p i r a t o r y discharge values a t r e s t i n g lung volume (a vs c). BLOOD GAS AND LUNG GAS ANALYSIS i) Maintained I n f l a t i o n with A i r At peak i n f l a t i o n , the mean values f o r the f r a c t i o n a l c o n c e n t r a t i o n of oxygen and carbon dioxide i n the lungs were 20.27% (+/-.30 S.E.M.) and .99% (+/- .36 S.E.M.) r e s p e c t i v e l y . At the end of the 1 hour p e r i o d o f maintained i n f l a t i o n on a i r , lung gas F02 decreased to 13.17% (+/- 1.19 S.E.M.) and lung gas FC02 i n c r e a s e d to 2.60% ( + /- .57 S.E.M.). When the pe r i o d of maintained lung i n f l a t i o n was extended, a t the end of 6 hours the f r a c t i o n a l c o n c e n t r a t i o n s of oxygen and carbon dioxide were 1L84% (+/- L42 S.E.M.) and 4.41 (+/- 1.41 S.E.M.) r e s p e c t i v e l y (see f i g u r e 11). A d d i t i o n a l measurements of lung gas FC02 values are d i s p l a y e d on tab l e 1. At peak, lung i n f l a t i o n , the mean a r t e r i a l PC02 value was 12.00 mmHg ( + /- 1.30 S.E.M.) and the mean corresponding pH value was 7.94 (+/- .13 S.E.M.). A f t e r 1 hour o f maintained lung i n f l a t i o n , a r t e r i a l PC02 i n c r e a s e d to a mean value of 26.00 mmHg (+/- 2.45 S.E.M.) and pH decreased to a mean value of 7.71 (+/-0.04 S.E.M.). When the maintained i n f l a t i o n p e r i o d was extended to 6 hours, the mean a r t e r i a l PC02 and pH values were 74.50 mmHg ( + /- 7.28 S.E.M.) and 7.12 ( + /- .03 S.E.M.) r e s p e c t i v e l y (see fi g u r e 12). A d d i t i o n a l a r t e r i a l PC02 values are l i s t e d i n table 1. ii ) Maintained I n f l a t i o n with Increased L e v e l s of Insp i r e d C02 P r i o r to C02 ad d i t i o n , the mean values f o r the f r a c t i o n a l c o n c e n t r a t i o n s o f oxygen and carbon dioxide i n the lung gas were 20.25% ( + /- .39 S.E.M.) and .78% ( + /- 0.55 S.E.M.) r e s p e c t i v e l y . At peak, i n f l a t i o n ( a f t e r a t l e a s t 5 minutes of pump v e n t i l a t i o n with 5% C02 i n a i r ) , the mean values f o r lung gas P02 and PC02 were 20.67% (+/- 0.20 S.E.M.) and 4.59% (+/- 0.28 S.E.M.) r e s p e c t i v e l y . A f t e r 1 hour o f maintained lung i n f l a t i o n on hypercapnic a i r , lung gas P02 decreased to 17.13% (+/- 1.53 S.E.M.) and lung gas PC02 i n c r e a s e d to 5.74% ( + /- 0.44 S.E.M.). 5 0 FIGURE 11 Lung Gas A n a l v s i s - M a i n t a i n e d I n f l a t i o n on A i r F r a c t i o n a l c o n c e n t r a t i o n o f l u n g gas oxygen and c a r b o n d i o x i d e (%) p r e s e n t i n the l u n g s a t peak i n f l a t i o n , 1 hour o f m a i n t a i n e d l u n g e l e v a t i o n and 6 hours o f m a i n t a i n e d e l e v a t i o n . Mean v a l u e s (+/- S.E.M.) n= 8 a n i m a l s denotes l u n g gas F 0 2 denotes l u n g gas F C 0 2 51 TABLE 1. Blood and Lung Gas Measurements Maintained I n f l a t i o n on A i r B a s e l i n e Peak I n f l a t i o n 150 sec. i n f l a t i o n 1 hour i n f l a t i o n 2 hour i n f l a t i o n 4 hour i n f l a t i o n 6 hour i n f l a t i o n PaC02 (mmHg) 12.00 (1.30) 12.00 (1.30) 26.00 (2.45) 45.25 (6.06) 60.88 (6.19) 74.50 (7.28) FAC02 (%) 1.08 (0.36) .99 (0.36) 1.19 (0.35) 2.60 (0.57) 4.14 (3.95) Maintained I n f l a t i o n on C02 PaC02 (mmHg) FAC02 (%) P r i o r to CO2 a d d i t i o n B a s e l i n e 33.14 (6.71) 0.78 (0.55) A f t e r 5. Min. on C02 Peak I n f l a t i o n 37.43 (4.84) 4.56 (0.28) 150 sec. I n f l a t i o n 1 hour i n f l a t i o n 47.42 (4.75) 5.74 (0.44) 2 hour i n f l a t i o n 53.80 (6.26) ' 4 hour i n f l a t i o n 61.40 (7.24) 6 hour i n f l a t i o n 70.60 (8.40) 10.26 (1.36) Mean Values (+/- S.E.M.) PaC02 FAC02 = p a r t i a l p r e ssure o f C02 i n a r t e r i a l blood = f r a c t i o n a l c o n c e n t r a t i o n o f C02 i n a l v e o l a r lung gas 52 FIGURE 12 B l o o d Gas A n a l y s i s - M a i n t a i n e d I n f l a t i o n on A i r P a r t i a l p r e s s u r e s (mmHg) o f ca r b o n d i o x i d e and pH v a l u e s o f the b l o o d a t l u n g i n f l a t i o n ; and 1,2,4, and 6 hours o f m a i n t a i n e d i n f l a t i o n . Mean v a l u e s (+/- S.E.M.) n= 10 a n i m a l s denotes a r t e r i a l pH denotes a r t e r i a l PC02 52a 53 When the pe r i o d o f maintained lung i n f l a t i o n was extended, a t the end o f 6 hours the f r a c t i o n a l c o n c e n t r a t i o n s of oxygen and carbon dioxide were 8.18% ( + /- 3.16 S.E.M.) and 10.26% ( + /- 1.36 S.E.M.) r e s p e c t i v e l y (see f i g u r e 13). P r i o r to C02 a d d i t i o n , the mean value f o r a r t e r i a l PC02 was 33.14 mmHg ( + /- 6.71 S.E.M.) and the mean corresponding pH value was 7.55 (+/- 0.14 S.E.M.). At peak, i n f l a t i o n (following 5 minutes of pump v e n t i l a t i o n with 5% C02 i n a i r ) , a r t e r i a l PC02 had i n c r e a s e d to 37.4 3 mmHg (+/- 4.84 S.E.M.) and the a r t e r i a l pH had decreased to 7.46 ( + /- 0.08 S.E.M.). A f t e r 1 hour of maintained i n f l a t i o n on hypercapnic a i r , a r t e r i a l PC02 had f u r t h e r i n c r e a s e d to 47.42 mmHg ( + /- 12.36 S.E.M.) and a r t e r i a l pH decreased to 7.35 (+/- 0.06 S.E.M.). When the maintained lung i n f l a t i o n p e r i o d was extended to 6 hours, a r t e r i a l PC02 had reached 70.60 mmHg (+/- 8.40 S.E.M.) and a r t e r i a l pH had decreased to 7.09 ( + /- 0.07 S.E.M.) (see f i g u r e 14). 54 FIGURE 13 Lung Gas A n a l y s i s - Maintained I n f l a t i o n on C02 F r a c t i o n a l c o n c e n t r a t i o n of oxygen and carbon d i o x i d e (%) prese n t i n the lung gas p r i o r to C02 a d d i t i o n , 5 minutes a f t e r C02 a d d i t i o n and a t 1 and 6 hours f o l l o w i n g maintained lung i n f l a t i o n on 5% C02 i n a i r . Mean values (+/- S.E.M.) n=10 animals ™ denotes lung gas F02 ^ denotes lung gas FC02 55 FIGURE 14 B l o o d Gas A n a l y s i s - M a i n t a i n e d I n f l a t i o n on C02 P a r t i a l p r e s s u r e s (mmHg) o f ca r b o n d i o x i d e and pH v a l u e s o f the b l o o d p r i o r t o C02 a d d i t i o n , 5 minutes a f t e r C02 a d d i t i o n and a t 1,2,4, and 6 hours o f m a i n t a i n e d i n f l a t i o n on 5% C02 i n a i r . Mean v a l u e s (+/- S.E.M.) n= 10 a n i m a l s denotes a r t e r i a l pH denotes a r t e r i a l PC02 56 RAPIDLY ADAPTING PULMONARY STRETCH RECEPTORS Figure 15 i l l u s t r a t e s the discharge p r o f i l e of a r a p i d l y adapting pulmonary s t r e t c h r e c e p t o r (RAR) during a maintained lung i n f l a t i o n stimulus. With the onset of lung i n f l a t i o n from f u n c t i o n a l r e s i d u a l lung volume (airway p r e s s u r e = 0 cmH20) to an e l e v a t e d lung volume (airway p r e s s u r e = 20 cmH20), the r e c e p t o r discharge r a t e i n c r e a s e s d r a m a t i c a l l y . The peak, i n f l a t i o n discharge r a t e i s 20 Hz and i t quickly decays to i t s baseline ( r e s t i n g lung volume) l e v e l o f 1 to 2 Hz within 3 seconds. We recorded from 6 RARs but no d e t a i l e d a n a l y s i s were c a r r i e d o u t on t h i s r e c e p t o r group. 57 FIGURE 15 Response of §_ R a p i d l y Adapting Pulmonary S t r e t c h  Receptor to S t a t i c Lung I n f l a t i o n PAW = airway pressure (0 to 10 cmH20) 58 DISCUSSION S e v e r a l i n t e r e s t i n g r e s u l t s have been pr e s e n t e d i n t h i s study which w i l l be e l a b o r a t e d on i n the following d i s c u s s i o n . Slowly adapting pulmonary s t r e t c h r e c e p t o r s recorded from i n t h i s study did not completely adapt to long term maintained i n c r e a s e s i n lung volume. Furthermore, n e i t h e r the discharge r a t e a t t a i n e d with s t a t i c lung i n f l a t i o n nor the discharge r a t e to which the r e c e p t o r s adapted a t 150 seconds of maintained i n f l a t i o n were a f f e c t e d by long term maintained lung i n f l a t i o n s with a i r . However, both the r a t e of r e c e p t o r a d a p t a t i o n and the response of the r e c e p t o r s to dynamic lung i n f l a t i o n s were a f f e c t e d by one hour of maintained lung i n f l a t i o n with a i r . Maintained lung i n f l a t i o n s with 5% C02 i n a i r r e s u l t e d i n an o v e r a l l decrease i n the l e v e l o f discharge of the slowly adapting pulmonary s t r e t c h r e c e p t o r s . D i s t i n c t i o n Between Low and High Threshold Slowly Adapting  Pulmonary S t r e t c h Receptors: Two populations o f slowly adapting pulmonary s t r e t c h r e c e p t o r s were r e c o r d e d from i n t h i s study; those t h a t e x h i b i t e d a t o n i c discharge a t f u n c t i o n a l r e s i d u a l c a p a c i t y (low t h r e s h o l d r e c e p t o r s ) and those t h a t only responded when lung volume exceeded FRC (high t h r e s h o l d r e c e p t o r s ) . In his r e c e n t review, P a i n t a l (198 3) has made a s i m i l a r d i s t i n c t i o n ; low t h r e s h o l d r e c e p t o r s are those t h a t f i r e during both i n s p i r a t i o n and e x p i r a t i o n and high t h r e s h o l d r e c e p t o r s are those t h a t f i r e only during i n s p i r a t i o n . Mammalian s t u d i e s have demonstrated t h a t most high t h r e s h o l d r e c e p t o r s are l o c a t e d i n the l a r g e r diameter intrapulmonary airways ( c h a r a c t e r i s t i c o f the p e r i p h e r a l p o r t i o n s of the lung) while low t h r e s h o l d r e c e p t o r s are more widely d i s t r i b u t e d throughout the pulmonary airways (Sant'Ambrogio and Sant'Ambrogio, 1980; P a i n t a l and Ravi, 1980). In t h i s study there was no attempt to l o c a l i z e the SARs. Owing to the high degree of morphological v a r i a t i o n between r e p t i l i a n and mammalian lungs (Romer and Parson, 1977), i t i s d i f f i c u l t to say whether or not the low and high t h r e s h o l d SARs t h a t were rec o r d e d from were i n f a c t anatomically d i s t i n c t populations. The data p r e s e n t e d i n t h i s study demonstrates t h a t both the low and high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s have the same c l a s s i c a l responses to changes i n lung volume. However, there appears to be some d i f f e r e n c e s i n t h e i r a d a p t a t i o n responses as w e l l as i n t h e i r s e n s i t i v i t i e s to i n c r e a s e d l e v e l s o f i n s p i r e d C02. LOW THRESHOLD SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS  Tonic Response to S t a t i c Lung I n f l a t i o n : As i l l u s t r a t e d i n f i g u r e 5, 1 hour of maintained r e s t i n g lung volume ( c o n t r o l protocol) had no e f f e c t on e i t h e r the peak discharge response to s t a t i c lung i n f l a t i o n or the r a t e to which the r e c e p t o r s adapted during the s h o r t term i n f l a t i o n stimulus (panel C). Furthermore, there was no change i n the a d a p t a t i o n 60 indices following one hour of maintained r e s t i n g lung volume (figure 6, c o n t r o l protocol). The c o n t r o l p r o t o c o l has s e r v e d to e s t a b l i s h t h a t maintaining the lungs a t r e s t i n g volume f o r a p e r i o d of 1 hour (with no c y c l i c v e n t i l a t i o n ) , had no e f f e c t on the s t a t i c i n f l a t i o n discharge response of the slowly adapting pulmonary s t r e t c h r e c e p t o r s . Therefore any changes observed i n t h i s s t a t i c i n f l a t i o n response a f t e r the lungs had been maintained a t an e l e v a t e d volume ( t e s t protocol) or a t an e l e v a t e d volume with 5% C02 i n a i r ( C 02-test p r o t o c o l ) , should be purely a f u n c t i o n o f the t e s t c o n d i t i o n . A f t e r 1 hour of maintained i n f l a t i o n with a i r ( t e s t p r o t o c o l ) , the slowly adapting pulmonary s t r e t c h r e c e p t o r s had adapted 80% from t h e i r peak, discharge value (figure 5). Most of t h i s a d a p t a t i o n o c c u r r e d within the f i r s t 2.5 minutes of i n f l a t i o n . The only, documentation of d i r e c t r e c o r d i n g s made from SARs over an extended p e r i o d of s u s t a i n e d s t i m u l a t i o n was done by Davenport e_t a l . , (1981) on t r a c h e a l s t r e t c h r e c e p t o r s i n dogs. They demonstrated t h a t a f t e r 1 hour of s u s t a i n e d s t i m u l a t i o n (by s t r e t c h i n g the t r a c h i a l i s muscle), the t r a c h e a l s t r e t c h r e c e p t o r s had adapted to within 30% to 80% o f t h e i r p r e - s t r e t c h values. They a l s o showed t h a t most of the a d a p t a t i o n o c c u r r e d wi t h i n the f i r s t 3 minutes of the muscle s t r e t c h i n g process. Thus r e s u l t s p r e s e n t e d i n t h i s study agree w e l l with those of Davenport e t a l . , 1981. The a d a p t a t i o n response of slowly adapting pulmonary s t r e t c h receptors is a characteristic common to most mechanoreceptors. The complete mechanism involved in receptor adaptation is not fully understood but i t is believed to have both a mechanical and an ionic basis. The mechanical basis of receptor adaptation pertains primarily to changes in the structural properties of the tissue in which the receptor sits (Patton, 1965). Davenport et. al.. (1981) have attributed the adaptation response of slowly adapting tracheal stretch receptors to changes in the mechanical properties of the trachealis muscle itself. Davenport et al.. (198D have stimulated the vagus nerve distally from the recording electrodes (antidromic stimulation) throughout the course of the muscle stretching process and found that this failed to alter the adaptation indices of these receptors. From these results, they concluded that stretch receptor adaptation was caused by changes in the mechanical properties of the tissue and was not an intrinsic property of the receptor memebrane itself. One cannot exclude the possibility of an ionically based receptor adaptation mechanism. In order to explain the ionic basis of receptor adaptation, one requires a basic knowledge of the mechanoreceptor stimulus modality. The mechanoreceptor acts as a transducer and in effect, transforms one form of energy into another. Deformations of the tissue in which the receptor sits are transformed into a graded generator (receptor) potential. The intensity of the generator potential is encoded by the spike encoder through modulation of the generator potential amplitude. 62 The spike encoder then tra n s f o r m s t h i s graded p o t e n t i a l i n t o a s e r i e s of frequency modulated a c t i o n p o t e n t i a l s (Patton, 1965). According to Grigg (1986), the i o n i c mechanism o f s t r e t c h r e c e p t o r a d a p t a t i o n has two p o s s i b l e s i t e s of a c t i o n ; a t the l e v e l o f the sensory ending (thus a f f e c t i n g the amplitude o f the gener a t o r p o t e n t i a l ) and a t the l e v e l of the spike encoder (by producing a p o s t - s p i k e decrease i n the membrane e x c i t a b i l i t y ) . Obviously any mechanism t h a t succeeds i n r e t u r n i n g the gene r a t o r p o t e n t i a l to i t s r e s t i n g value w i l l c o n t r i b u t e to r e c e p t o r adaptation. The proposed i o n i c mechanisms are s a i d to involve c a t i o n - g a t e d channels which, when a c t i v a t e d , s e r v e to r e p o l a r i z e the r e c e p t o r ending (Grigg, 1986). The most l i k e l y s i t e of i o n i c r e c e p t o r a d a p t a t i o n i s a t the l e v e l o f the spike encoder. Again, the membrane i s thought to be r e p o l a r i z e d by an e l e c t r o g e n i c Na + pump t h a t i s s t i m u l a t e d by the Na+ i n f l u x a t the onset of the preceeding a c t i o n p o t e n t i a l . Figure 5 ( t e s t p r o tocol) demonstrates t h a t the peak i n f l a t i o n discharge r a t e i n response to s t a t i c lung i n f l a t i o n was not s i g n i f i c a n t l y a f f e c t e d by the hour o f maintained lung i n f l a t i o n , nor was the s t e a d y - s t a t e discharge r a t e to which the r e c e p t o r s e v e n t u a l l y adapted. However, there was a d i f f e r e n c e i n the a d a p t a t i o n i n d i c e s c a l c u l a t e d before and a f t e r the hour o f maintained lung i n f l a t i o n (figure 6, t e s t protocol). Since the r e c e p t o r s showed the same discharge response to s t a t i c lung i n f l a t i o n before and a f t e r the c o n t r o l run, the d i f f e r e n c e i n the a d a p t a t i o n i n d i c e s seen i n the t e s t r u n must be a f u n c t i o n o f the 63 adaptive process that occured during the maintained lung inflation period. These differences in the receptor adaptation indices may have a mechanical basis. If the receptor adaptation observed over the hour long maintained lung inflation period is attributable to the ensuing changes in the structural properties of the pulmonary tissue surrounding the stretch receptor (Davenport eJt si . , 1981), then perhaps the more rapid rate of receptor adaptation following maintained inflation is due tp a perpetuation of this mechanical phenomenon. After 1 hour of maintained lung inflation with 5% C02 in air the slowly adapting pulmonary stretch receptors had adapted 88% (figure 5, C02-test protocol). Following the period of maintained lung inflation with 5% C02 in air, the peak discharge response of the SARs to the Increase in lung volume was significantly lower than with the same inflation stimulus prior to C02-addition, as was the steady-state level of discharge to which the receptors eventually adapted during the short term (150 seconds) inflation stimulus. Furthermore, the adaptation Indices following the period of maintained lung inflation with 5% C02 were larger than those prior to this period of maintained lung inflation (figure 6, C02-test protocol. These results show that the levels of receptor discharge attained throughout the C02-test protocol following maintained inflation with 5% C02 were consistantly lower than those prior to the addition of C02. Interestingly, the adaptation indices following maintained i n f l a t i o n with 5% C02 were not g r e a t e r than those following maintained i n f l a t i o n with a i r . That SAR discharge i s i n some way modified by i n c r e a s e d l e v e l s o f i n s p i r e d C02 has been documentated i n mammals (Mustafa and Purves, 1972; Sant'Ambrogio a l . , 1974; B a r t l e t t and Sant'Ambrogio, 1976; Coleridge e_t a l . , 1978; M i t c h e l l e t a l . , 1980,) as w e l l as i n r e p t i l e s (Milsom and Jones, 1976; Fedde e_t a l . , 1977; Jones and Milsom, 1979). The r e s u l t s p resented i n t h i s study are i n agreement with the above authors. U n f o r t u n a t e l y , the r e s u l t s from t h i s study give no f u r t h e r i n s i g h t i n t o the mechanism i n v o l v e d i n the C02-induced decrease i n SAR discharge. Phasic Response to S t a t i c Lung I n f l a t i o n and D e f l a t i o n ; The slowly adapting pulmonary s t r e t c h r e c e p t o r s t h a t were recorded from i n t h i s study e x h i b i t e d s i m i l a r discharge c h a r a c t e r i s t i c s to those r e p o r t e d i n mammals (Miserocchi and Sant'Ambrogio, 1974; B a r t l e t t e_t a l . , 1976; Davenport e_t a l . , 1981; Sant'Ambrogio e_t a l . , 198 3), i n t u r t l e s (Jones and Milsom, 1979) and i n lunged f i s h (Milsom and Jones, 1985). As i l l u s t r a t e d i n f i g u r e 5, an i n c r e a s e i n lung volume r e s u l t e d i n a dramatic i n c r e a s e i n SAR discharge followed by a slow r a t e o f adaptation. The i n i t i a l "overshoot" i n discharge i n response to sudden lung i n f l a t i o n i s a r a t e dependent phenomenon known as the on-response. Although the maximum discharge r a t e achieved by lung i n f l a t i o n i s a f u n c t i o n of both the volume and the r a t e of lung f i l l i n g , the l e v e l of a d a p t a t i o n a t t a i n e d with prolonged lung 65 i n f l a t i o n i s independent o f the i n f l a t i o n r a t e (Milsom and Jones, 1985). When the lung i n f l a t i o n stimulus was removed (during d e f l a t i o n to r e s t i n g lung volume), the r e c e p t o r discharge ceased b r i e f l y and then r e t u r n e d to i t s normal discharge r a t e f o r t h a t p a r t i c u l a r lung volume (figure 3b). This phenomenon, known as the o f f - r e s p o n s e , i s thought to be due to an exaggerated h y p e r p o l a r i z a t i o n of the r e c e p t o r g e n e r a t o r p o t e n t i a l (Patton, 1965). The o f f - r e s p o n s e i s a uniform c h a r a c t e r i s t i c o f most v e r t e b r a t e mechanoreceptors including those found i n nonpulmonary t i s s u e s (muscle, gut and b a r o r e c e p t o r s (Patton, 1965) and ph o t o r e c e p t o r s (Kandel and Schwartz, 1981)). Phasic Response to Dynamic Lung I n f l a t i o n s ; Adrian (1933) f i r s t d e s c r i b e d the responses of slowly adapting pulmonary s t r e t c h r e c e p t o r s as being volume-related such t h a t spontaneous i n c r e a s e s and decreases i n lung volume r e s u l t e d i n r e s p e c t i v e i n c r e a s e s and decreases i n discharge. As i l l u s t r a t e d i n f i g u r e 7, pump v e n t i l a t i o n superimposed upon an e l e v a t e d lung volume r e s u l t e d i n s i g n i f i c a n t l y g r e a t e r l e v e l s o f peak i n s p i r a t o r y and en d - e x p i r a t o r y l e v e l s of discharge than did pump v e n t i l a t i o n a t r e s t i n g lung volume. These r e s u l t s agree w e l l with those of B a r t l e t t e t a l . , (1976), Muza and F r a z i e r , (1983) and Milsom and Jones, (1985). The low t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s t h a t were recorded from i n t h i s study c l e a r l y demonstrated a dynamic o f f - r e s p o n s e . At both r e s t i n g and e l e v a t e d lung volumes, the mean e n d - e x p i r a t o r y discharge values were lower than the p r e - i n f l a t i o n (baseline) discharge values even though the corresponding lung volumes were the same. This dynamic o f f - r e s p o n s e has p r e v i o u s l y been shown to be a r a t e s e n s i t i v e phenomenon probably due to an exaggerated h y p e r p o l a r i z a t i o n of the r e c e p t o r g e n e r a t o r p o t e n t i a l (Patton, 1965; Milsom and Jones, 1985). A f t e r 1 hour of maintained r e s t i n g lung volume, the baseline ( p r e - i n f l a t i o n ) , peak i n s p i r a t o r y and en d - e x p i r a t o r y discharge r a t e s a t both e l e v a t e d and r e s t i n g lung volumes remained unchanged from the corresponding r a t e s p r i o r to maintained r e s t i n g lung volume (figure 7, c o n t r o l protocol). The absolute number o f impulses f i r e d during the i n s p i r a t o r y phase p r i o r to and following the 1 hour p e r i o d o f maintained r e s t i n g lung volume were l e s s c o n s i s t e n t (figure 8, c o n t r o l protocol). At both e l e v a t e d and r e s t i n g lung volumes, the mean number o f impulses f i r e d during i n s p i r a t i o n was reduced by approximatley 8% following the 1 hour p e r i o d of maintained r e s t i n g lung volume. R e s u l t s from the c o n t r o l p r o t o c o l have s e r v e d to e s t a b l i s h t h a t the r a t e o f SAR discharge during the ba s e l i n e , peak i n s p i r a t o r y and e n d - e x p i r a t o r y p o r t i o n s of the v e n t i l a t o r y c y c l e were not a f f e c t e d by maintaining the lungs a t r e s t i n g lung volume f o r a perio d o f 1 hour. Furthermore, the abso l u t e number of impulses f i r e d during the e x p i r a t o r y phase o f the v e n t i l a t o r y c y c l e did not change with the 1 hour p e r i o d of maintained r e s t i n g lung volume. Any changes observed i n the above components of the cyc l e following 1 hour of maintained lung i n f l a t i o n with a i r ( t e s t protocol) or 1 hour o f maintained lung i n f l a t i o n with 5% C02 i n a i r (C02-test protocol) should be a f u n c t i o n of the t e s t condition. However, care must be taken i n i n t e r p r e t i n g data concerning the number o f impulses f i r e d during the i n s p i r a t o r y phase of the v e n t i l a t o r y c y c l e . A f t e r 1 hour of maintained i n f l a t i o n with a i r , the dynamic discharge r a t e s of the slowly adapting pulmonary s t r e t c h r e c e p t o r s were s i g n i f i c a n t l y reduced during peak i n s p i r a t i o n and en d - e x p i r a t i o n a t both e l e v a t e d and r e s t i n g lung volumes (figure 7, t e s t p rotocol). A d d i t i o n a l l y , t h e r e was a decrease i n the absol u t e number of impulses f i r e d during both the i n s p i r a t o r y and e x p i r a t o r y phases of the pump v e n t i l a t i o n c y c l e (figure 8, t e s t protocol). There was no d i f f e r e n c e i n the baseline discharge r a t e s of these r e c e p t o r s following the pe r i o d of maintained lung i n f l a t i o n . These r e s u l t s i n d i c a t e t h a t the hour o f maintained i n f l a t i o n on a i r produced a decrease i n both the r a t e of discharge and i n the absolute number o f impulses f i r e d during the i n s p i r a t o r y and e x p i r a t o r y p o r t i o n s of the pump v e n t i l a t i o n c y c l e a t both r e s t i n g and e l e v a t e d lung volumes. However, from these data i t i s d i f f i c u l t to determine which f a c t o r was a f f e c t e d more, the r a t e of r e c e p t o r discharge or the absolute number of impulses f i r e d . A f t e r 1 hour of maintained i n f l a t i o n with 5% C02 i n a i r , the baseline and the dynamic (peak i n s p i r a t o r y and end-expiratory) 68 discharge r a t e s were a l s o s i g n i f i c a n t l y reduced a t both e l e v a t e d and r e s t i n g lung volumes (figure 7, C02-test). There was a l s o a dramatic decrease i n the number of impulses f i r e d during the i n s p i r a t o r y phase of the v e n t i l a t o r y c y c l e (figure 8, C 0 2 - t e s t protocol). The decreases i n the discharge r a t e s during the pump v e n t i l a t i o n were g r e a t e r than those following maintained i n f l a t i o n with a i r as were the decreases i n the abs o l u t e number of impulses o c c u r i n g during the i n s p i r a t o r y phase o f the cy c l e . As p r e v i o u s l y demonstrated by Jones and Milsom (1979), t u r t l e pulmonary s t r e t c h r e c e p t o r s show a dramatic decrease i n t h e i r s e n s i t i v i t y to lung i n f l a t i o n with i n c r e a s e d l e v e l s of i n s p i r e d C02. I t i s not p o s s i b l e to determine whether C02 had a more pronounced e f f e c t on the r a t e of change of discharge or on the o v e r a l l l e v e l o f discharge r a t e . These r e s u l t s are a l s o i n c l o s e agreement with those p r e s e n t e d by Mustfa and Purves (1972) and Bradley e t a l . , (1974) f o r mammals. HIGH THRESHOLD SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS  Tonic Response to S t a t i c Lung I n f l a t i o n : As i l l u s t r a t e d i n f i g u r e 9, 1 hour of maintained r e s t i n g lung volume ( c o n t r o l p r o t o c o l ) had no e f f e c t on e i t h e r the peak response to s t a t i c lung i n f l a t i o n or the r a t e to which the r e c e p t o r s adapted during the s h o r t term i n f l a t i o n stimulus. Furthermore, the r a t e a t which the r e c e p t o r s adapted to lung volume was unchanged by the 1 hour perio d of maintained r e s t i n g lung volume. The c o n t r o l p r o t o c o l has t h e r e f o r e e s t a b l i s h e d t h a t maintaining the lungs a t r e s t i n g volume f o r 1 hour had no e f f e c t on the s t a t i c i n f l a t i o n response of the high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s . Therefore, any changes observed i n t h i s s t a t i c i n f l a t i o n response a f t e r the lungs had been maintained a t an e l e v a t e d volume ( t e s t p r o tocol) or a t an e l e v a t e d volume with 5% C02 i n a i r (C02-test p r o t o c o l ) , should be purely a f u n c t i o n o f the t e s t condition. A f t e r 1 hour of maintained i n f l a t i o n with a i r , the high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s had adapted to 75% o f t h e i r peak discharge l e v e l . A l l of t h i s a d a p t a t i o n o c c u r r e d within the f i r s t 2.5 minutes of lung i n f l a t i o n . The peak discharge r a t e i n response to s t a t i c lung i n f l a t i o n was not s i g n i f i c a n t l y a f f e c t e d by the hour o f maintained lung i n f l a t i o n , nor was the s t e a d y - s t a t e discharge r a t e to which the r e c e p t o r s e v e n t u a l l y adapted (figure 9, t e s t protocol). The r a t e of r e c e p t o r a d a p t a t i o n did not seem to be a f f e c t e d by the t e s t condition. Since the values of the a d a p t a t i o n i n d i c e s f o r t h i s p o p u l a t i o n were highly v a r i a b l e (probably due to the small sample size) , they are not presented. The high t h r e s h o l d SARs did not adapt as much as the low t h r e s h o l d SARs (25% vs 80%) during the hour of maintained lung i n f l a t i o n with a i r . This decrease i n a d a p t a t i o n s e n s i t i v i t y may be an i n h e r e n t c h a r a c t e r i s t i c o f the r e c e p t o r s themselves (ionic adaptation), or i t may be a t t r i b u t a b l e to changes i n the p h y s i c a l p r o p e r t i e s o f the t i s s u e s surrounding the r e c e p t o r s (mechanical adaptation). I f one may assume t h a t there i s a s i g n i f i c a n t 70 mechanical b a s i s f o r the a d a p t a t i o n phenomenon, then perhaps the high and low t h r e s h o l d SARs rec o r d e d from i n t h i s study were s i t u a t e d i n d i f f e r e n t t i s s u e types which i n t u r n e x h i b i t e d d i f f e r e n t responses to volume induced p e r t u r b a t i o n s . If t h i s assumption i s v a l i d , then these r e c e p t o r s may i n f a c t be anatomically d i s t i n c t populations (as o r i g i n a l l y suggested by PaintaL- 198 3). Since data regarding the p r e c i s e l o c a t i o n of the SARs rec o r d e d from i n t h i s study were not c o l l e c t e d , t h i s hypothesis must remain s p e c u l a t i v e . A f t e r 1 hour of maintained i n f l a t i o n with 5% C02 i n a i r , the high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s had adapted 45% (figure 9, C 0 2 - t e s t protocol). The r a t e of discharge a t t a i n e d a f t e r 1 hour of maintained i n f l a t i o n with 5% C02 i n a i r was s i g n i f i c a n t l y lower than t h a t a t t a i n e d a f t e r 2.5 minutes of lung i n f l a t i o n with a i r (panel A). Following the pe r i o d o f maintained lung i n f l a t i o n with 5% C02 i n a i r , the peak discharge response of the SARs to the i n c r e a s e i n lung volume was s i g n i f i c a n t l y lower than with the same i n f l a t i o n stimulus p r i o r to C02 a d d i t i o n , as was the s t e a d y - s t a t e l e v e l o f discharge to which the r e c e p t o r s e v e n t u a l l y adapted during the s h o r t term (150 seconds) maintained i n f l a t i o n stimulus. The decrease i n discharge over the 1 hour perio d of maintained lung i n f l a t i o n t h a t was due to the i n c r e a s e d l e v e l s of i n s p i r e d C02 can be estimated as follows: the discharge r a t e a t 60 minutes of maintained lung i n f l a t i o n with a i r minus the discharge r a t e a t 60 minutes of lung i n f l a t i o n with 5% C02 i n a i r . For the high t h r e s h o l d SARs, the decrease i n discharge due to the i n c r e a s e d l e v e l s of i n s p i r e d C02 (25%) was much g r e a t e r than t h a t c a l c u l a t e d f o r the low t h r e s h o l d SARs (4%). These es t i m a t e s i n d i c a t e t h a t the high t h r e s h o l d SARs were much more s e n s i t i v e to i n c r e a s e d l e v e l s of i n s p i r e d C02 than were the low th r e s h o l d SARs. Phasic Responses to S t a t i c Lung I n f l a t i o n and D e f l a t i o n : Like the low t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s , these high t h r e s h o l d SARs a l s o e x h i b i t e d a r a t e s e n s i t i v e on-response. As i l l u s t r a t e d i n f i g u r e 9, an i n c r e a s e i n lung volume r e s u l t e d i n a dramatic i n c r e a s e i n SAR discharge followed by a slow r a t e of adaptation. When the lung i n f l a t i o n stimulus was removed (during d e f l a t i o n to r e s t i n g lung volume), these r e c e p t o r s ceased t h e i r discharge r a t e and only f i r e d o c c a s i o n a l l y a t r e s t i n g lung volume and as such did not demonstrate an o f f - r e s p o n s e . Phasic Response to Dynamic Lung I n f l a t i o n s : As i l l u s t r a t e d i n f i g u r e 10, pump v e n t i l a t i o n superimposed upon the e l e v a t e d lung volume r e s u l t e d i n s i g n i f i c a n t l y g r e a t e r l e v e l s of peak i n s p i r a t o r y and en d - e x p i r a t o r y l e v e l s of discharge than did pump v e n t i l a t i o n a t r e s t i n g lung volume. These r e s u l t s agree w e l l with those of B a r t l e t t et_ a l . , (1976); Muza and F r a z i e r (198 3) and Milsom and Jones (1985). Unlike the low th r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s , i n most 72 cases these high t h r e s h o l d SARs did not demonstrate a s i g n i f i c a n t dynamic o f f - r e s p o n s e a t e l e v a t e d lung volume. A f t e r 1 hour of maintained i n f l a t i o n a t r e s t i n g lung volume, and 1 hour of maintained i n f l a t i o n a t e l e v a t e d lung volume, the baseline ( p r e - i n f l a t i o n ) , peak i n s p i r a t o r y and en d - e x p i r a t o r y discharge r a t e s a t both e l e v a t e d and r e s t i n g lung volumes remained unchanged from the corresponding r a t e s p r i o r to the periods o f lung volume maintenance (figure 10, c o n t r o l and t e s t r p r o t o c o l s ) . Maintained lung i n f l a t i o n with 5% C02 only a f f e c t e d the baseline discharge r a t e following the lung i n f l a t i o n period. These r e s u l t s i n d i c a t e t h a t n e i t h e r the maintained lung i n f l a t i o n stimulus nor the hypercapnic stimulus had an e f f e c t on the dynamic s e n s i t i v i t y of the high t h r e s h o l d slowly adapting pulmonary s t r e t c h r e c e p t o r s . Increased l e v e l s o f i n s p i r e d C02 did however serve to reduce the o v e r a l l l e v e l o f discharge r a t e (as seen by the changes i n the baseline values). Why the C02-induced decrease i n the o v e r a l l l e v e l o f discharge was not r e f l e c t e d i n corresponding decreases i n the peak i n s p i r a t o r y and end - e x p i r a t o r y discharge values i s unknown. BLOOD AND LUNG GAS ANALYSIS Since the n a t u r a l b r e a t h i n g p a t t e r n o f t u r t l e s i s p e r i o d i c (McCutcheon, 194 3), these animals tend to show large v a r i a t i o n s i n a r t e r i a l P02 and PC02 l e v e l s during normal i n t e r m i t t e n t breathing (Lefant e_t a l . , 1970) as w e l l as during extended periods o f d i v i n g (Jackson and S i l v e r b l a t t , 1974). The magnitude of these v a r i a t i o n s are f u r t h e r a f f e c t e d by i n t r a c a r d i a c blood shunting a s s o c i a t e d with the periods of b r e a t h holding (Burggren e t a l . , 1978). The a b i l i t y to p r e f e r e n t i a l l y p e r f u s e the systemic or the pulmonary c i r c u l a t i o n i s a c h a r a c t e r i s t i c of the physiology o f these animals and i s p o s s i b l e p r i m a r i l y because of the incomplete d i v i s i o n of the c a r d i a c v e n t r i c l e (Romer and Parsons, 1977). Because of the i n t e r m i t t e n t pulmonary p e r f u s i o n , i t i s not s u r p r i s i n g t h a t measurements of blood gas tensions do not always c l o s e l y follow measurements of the f r a c t i o n a l c o n c e n t r a t i o n s of gases i n the lung (Burggren and Shelton, 1979). The primary aim o f measuring a r t e r i a l PC02 and pH l e v e l s i n t h i s study was to monitor to metabolic s t a t e of the animal throughout the experimental p r o t o c o l , p a r t i c u l a r l y during the periods of maintained lung i n f l a t i o n . The measurements f o r a r t e r i a l PC02 and lung gas FC02 and F02 obtained a f t e r 1 hour of maintained lung i n f l a t i o n s with a i r ( f i g u r e s 11 and 12) e x h i b i t e d the same trends as r e s u l t s documented by Burggren and Shelton (1979) on the same species o f t u r t l e , Pseudemvs s c r i p t a during an hour long v o l u n t a r y dive. However, the PC02 and FC02 values p r e s e n t e d i n t h i s study were c o n s i s t a n t l y lower than those r e p o r t e d by Burggren and Shelton (1979) due to the r e l a t i v e hypocapnia induced by the pump v e n t i l a t i o n sequences p r i o r to commencement of the p r o t o c o l and the lower metabolism of the p i t h e d animals. In any case, r e s u l t s from t h i s study show t h a t during the hour long maintained i n f l a t i o n p e r i o d there were 74 i n c r e a s e s i n a r t e r i a l PC02 and corresponding decreases i n a r t e r i a l pH (figure 12) as w e l l as i n c r e a s e s and decreases i n the f r a c t i o n a l c o n c e n t r a t i o n of lung gas C02 and 02 r e s p e c t i v e l y (figure ID. The f r a c t i o n a l c o n c e n t r a t i o n o f C02 p r e s e n t a t the r e c e p t o r s i t e s during maintained lung i n f l a t i o n with a i r was within the same range as t h a t of an animal undergoing a one hour v o l u n t a r y dive (Burggren and Shelton, 1979). As a r e s u l t , the FC02 p r e s e n t a t the r e c e p t o r s i t e s during maintained lung i n f l a t i o n (or during maintained r e s t i n g lung volume) was within the p h y s i o l o g i c a l range experienced by the animal under n a t u r a l conditions. CONTRIBUTION OF C02 TO PULMONARY STRETCH RECEPTOR ADAPTATION Increased l e v e l s of i n s p i r e d C02 are known to reduce the o v e r a l l l e v e l o f discharge of slowly adapting pulmonary s t r e t c h r e c e p t o r s i n t u r t l e s . More s p e c i f i c a l l y , Jones and Milsom (1979) have shown t h a t the a d m i n i s t r a t i o n of 5% C02 (in air) and 10% C02 (in a i r ) r e s u l t i n r e s p e c t i v e 35% and 45% r e d u c t i o n s of both peak i n s p i r a t o r y and e n d - e x p i r a t o r y discharge. The a l i n e a r i t y of t h i s response demonstrates t h a t the SARs are more s e n s i t i v e to l e v e l s o f C02 t h a t might be experienced by the animal under normal p h y s i o l o g i c a l c o n d i t i o n s (such as during an extended breathhold or during a dive). During an extended breathhold pe r i o d , metabolic C02 i s s t o r e d not only i n the blood and t i s s u e s o f these animals, but i n the lung gas as w e l l (for review see 75 Shelton et al.. , 1986). As a r e s u l t , as the breathhold progresses, the amount of C02 in the v i c i n i t y of the receptor s i t e s w i l l increase. The r e s u l t s of Jones and Milsom (1979) in combination with the r e s u l t s presented i n this study allow us to predict the contribution of accumulated metabolic (lung gas) C02 to the adaptation response of the slowly adapting pulmonary stretch receptors seen during the 1 hour period of maintained lung i n f l a t i o n with a i r . Low Threshold Slowly Adapting Pulmonary Stretch Receptors; Estimates of the contribution of the accumulated metabolic (lung gas) C02 to the adaptation response seen i n the low threshold pulmonary stretch receptors during the 1 hour of maintained lung i n f l a t i o n with a i r are graphically i l l u s t r a t e d in figure 16. The difference between the f r a c t i o n a l concentration of C02 present i n the lungs at the onset of lung i n f l a t i o n and at the end of short term (150 seconds) maintained lung i n f l a t i o n was 2.20% (table 1). Based on the r e s u l t s of Jones and Milsom (1979), the difference between 0.99% C02 and 1.19% C02 w i l l r e s u l t in a 2% decrease i n SAR discharge. Therefore, i n t h i s study, approximately 1% (2% of the 36% decrease) of the t o t a l reduction in discharge seen during the adaptation response to the short term maintained lung i n f l a t i o n was due to the accumulated metabolic C02 and the remainder (35%) was due to receptor 76 FIGURE 16 C o n t r i b u t i o n o f C02 to Pulmonary S t r e t c h R e c eptor  A d a p t a t i o n Response o f LOW THRESHOLD SARs Bar 1 mean peak i n f l a t i o n Bar 2 mean d i s c h a r g e r a t e l u n g i n f l a t i o n on a Bar 3 a i r mean d i s c h a r g e r a t e l u n g i n f l a t i o n w i t h Bar 3 C02 mean d i s c h a r g e r a t e l u n g i n f l a t i o n w i t h d i s c h a r g e r a t e a t 150 seconds o f m a i n t a i n e d i r a t 60 minutes o f m a i n t a i n e d a i r a t 60 minutes o f m a i n t a i n e d 5% C02 i n a i r ?6a Contribution of CO2 to Pulmonary Stretch Receptor Adaptati 1 2 vent.—1 • vent I • vent. vent.-100 i 80 1 6 0 1 4 0 1 20H P A C 0 2 = . 9 9 % P A C 0 2 = 1.19% adaptation CO2 measured CO2 calculated P A C O 2 = 2.60% P A c o 2 = 5.70% 3 a i r 3 CO2 77 adaptation. S i m i l a r l y , the difference between the f r a c t i o n a l concentration of C02 present i n the lungs at the end of the short term maintained i n f l a t i o n was 1.41%. Extrapolating from the res u l t s of Jones and Milsom (1979) where the difference between 1.19% and 2.60% C02 resulted i n a 10% decrease i n receptor discharge, one can estimate that i n this study, s l i g h t l y less than 2% (10% of the 16% decrease) of the further decrease i n receptor discharge over the 1 hour period of maintained i n f l a t i o n was due to accumulated metabolic C02 and the remainder (14%) was a r e s u l t of receptor adaptation. The difference i n the l e v e l of discharge at the end of 1 hour of maintained i n f l a t i o n with 5% C02 and at the end of 1 hour of maintained i n f l a t i o n with a i r (a 4% decrease), can be attributed to the difference i n the f r a c t i o n a l concentration of C02 i n the lung gas at thi s point i n the protocol (3.1%). These r e s u l t s indicate that the low threshold SARs recorded from i n t h i s study are mildly sensitive to increased l e v e l s of lung gas C02 (from 0.99% to 5.7%). HIGH THRESHOLD SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS I have already established that the high threshold SARs are more sensitive than the low threshold SARs are to increased levels of inspired C02. Since I have calculated the C02 contribution to the decrease i n discharge i n the low threshold 78 SARs based on the receptor C02 s e n s i t i v i t y relationships reported by Jones and Milsom (1979), these relationships cannot be applied to predict the C02 dependent reduction i n discharge i n the high threshold SARs. Despite the a l i n e a r i t y of the response s e n s i t i v i t i e s of SARs to increased lev e l s of C02, a l l predictions for the high threshold SARs w i l l be based on measured differences in discharge between rates seen at the end of 1 hour maintained i n f l a t i o n with a i r (test protocol) and at the end of 1 hour maintained i n f l a t i o n with 5% C02 i n a i r (C02-test protocol). As previously i l l u s t r a t e d - i n figure 9 (test protocol), there was no difference between the discharge rates at the end of the short term (150 seconds) maintained i n f l a t i o n period and at the end of the long term (1 hour) maintained i n f l a t i o n period. This re l a t i o n s h i p i s also i l l u s t r a t e d in figure 17. The difference in discharge at the end of long term maintained i n f l a t i o n with a i r and at the end of long term maintained i n f l a t i o n with 5% C02 in a i r (a 25% decrease) can be attributed to the differences in the f r a c t i o n a l concentration of C02 i n the lung gas of these animals at this point i n the protocol (3.1% C02) (see figure 17). Since 3.1% C02 resulted in a 25% reduction in discharge, the difference i n FAC02 between peak i n f l a t i o n and 150 seconds of maintained i n f l a t i o n (0.20% C02) should r e s u l t in a 6% reduction (25% of the 25% reduction) i n the o v e r a l l l e v e l of discharge. Therefore, 6% of the reduction i n discharge seen with short term maintained i n f l a t i o n was att r i b u t a b l e to the accumulated metabolic (lung gas) C02, and the remainder (19%), was due to 79 FIGURE 17 C o n t r i b u t i o n o f C02 t o Pulmonary S t r e t c h R e c eptor  A d a p t a t i o n Response o f HIGH THRESHOLD SARs Bar 1 mean peak, i n f l a t i o n d i s c h a r g e r a t e Bar 2 mean d i s c h a r g e r a t e a t 150 seconds o f m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r Bar 3 a i r mean peak i n f l a t i o n a t 60 minutes o f m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r Bar 3 C02 mean peak i n f l a t i o n a t 60 minutes o f m a i n t a i n e d l u n g i n f l a t i o n w i t h 5% C02 i n a i r 79a r—V-i 1 " 1 1—V~I — V - — V -< LU rx •s 80-e U J 60" CD or < ^ 40" to Q 20-0-F A C 0 2 = . 9 9 % ^^^^^^^ : HZ A D A P T A T I O N | | C0 2 M E A S U R E D M C0 2 C A L C U L A T E D F A C 0 2 = 1.19% F A C 0 2 = 2.60* F A C 0 2 = 5.70* 3 AIR 3 C O . 80 r e c e p t o r a d a p t a t i o n . The degree o f d i s c h a r g e r e d u c t i o n due t o C02 d u r i n g the s h o r t term m a i n t a i n e d i n f l a t i o n (6%) i s l i k e l y to be u n d e r e s t i m a t e d . As demonstrated by Jones and Milsom (1979), i n c r e a s e d l e v e l s o f i n s p i r e d C02 have the g r e a t e s t e f f e c t on SAR d i s c h a r g e a t the lower ( p h y s i o l o g i c a l ) l e v e l s . I n any c a s e , f i g u r e s 16 and 17 demonstrate t h a t a l t h o u g h low t h r e s h o l d SARs showed a l a r g e r a d a p t a t i o n r e sponse over 1 hour o f m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r t han the h i g h t h r e s h o l d SARs d i d , the h i g h t h r e s h o l d SARs were more s e n s i t i v e to i n c r e a s e d l e v e l s o f l u n g gas C02. EFFECT OF MINOR CHANGES IN. LUNG VOLUME ON. SLOWLY ADAPTING  PULMONARY STRETCH RECEPTOR DISCHARGE VALUES; The p r e s e n t s t u d y was performed on p i t h e d t u r t l e s u n d e r g o i n g a r t i f i c i a l l y i n d u c e d changes i n l u n g volume. Data g e n e r a t e d from t h i s s t u d y i s v e r y u s e f u l f o r f u r t h e r u n d e r s t a n d i n g the a d a p t a t i o n r e sponse of s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s to m a i n t a i n e d changes i n l u n g volume, as w e l l as t h e i r r e sponse t o s t a t i c and dynamic changes i n l u n g volume f o l l o w i n g the p e r i o d o f a d a p t a t i o n . F u r t h e r i n s i g h t has a l s o been g a i n e d i n t o the e f f e c t o f i n c r e a s e d l e v e l s o f i n s p i r e d C02 on the above r e s p o n s e s o f SARs. From the s e d a t a I have been a b l e t o e s t i m a t e the r e l a t i v e c o n t r i b u t i o n o f accumulated m e t a b o l i c C02 t o the a d a p t a t i o n p r o c e s s . E x t e n d i n g the a p p l i c a t i o n o f the r e s u l t s o b t a i n e d from the low t h r e s h o l d s l o w l y a d a p t i n g pulmonary s t r e t c h 81 r e c e p t o r s , one can e s t i m a t e the degree o f r e c e p t o r a d a p t a t i o n t h a t may o c c u r d u r i n g a 1 hour b r e a t h h o l d p e r i o d i n a normal ( i n t a c t ) t u r t l e . F i g u r e 18 i s a g r a p h i c a l r e p r e s e n t a t i o n o f the d i s c h a r g e r a t e a t the end o f the 1 hour p e r i o d p l o t t e d a g a i n s t l u n g volume f o r a h y p o t h e t i c a l 500 gram t u r t l e . Lung volume a t r e s t i n g l e v e l s ( f u n c t i o n a l r e s i d u a l c a p a c i t y ) was c a l c u l a t e d as 12 ml/100 grams ( J a c k s o n , 1971; Milsom, 1975). For the h y p o t h e t i c a l 500 gram t u r t l e , r e s t i n g l u n g volume i s 60 mis. 1 hour o f m a i n t a i n e d r e s t i n g l u n g volume w i l l r e s u l t i n an average d i s c h a r g e r a t e o f 36% o f peak (from f i g u r e 5, c o n t r o l p r o t o c o l ) . T h i s i s i l l u s t r a t e d as p o i n t A i n f i g u r e 18. For the h y p o t h e t i c a l t u r t l e , e l e v a t e d l u n g volume was c a l c u l a t e d as r e s t i n g l u n g volume p l u s the volume produced by a s t a t i c l u n g i n f l a t i o n o f 10 cmH20. From the s t a t i c c o m p l i a n c e c u r v e s o f V i t a l i s and Milsom (1986), one can dete r m i n e t h a t the average volume produced by a p r e s s u r e o f 10 cmH20 i s 8.4 ml/100 grams. T h e r e f o r e , the m a i n t a i n e d e l e v a t e d l u n g volume i n our h y p o t h e t i c a l t u r t l e i s 102 mis. A f t e r 1 hour o f m a i n t a i n e d e l e v a t e d l u n g volume, the average d i s c h a r g e o f the SARs w i l l be 44% o f peak (from f i g u r e 5, t e s t p r o t o c o l ) . T h i s i s i l l u s t r a t e d as p o i n t B i n f i g u r e 18. When t u r t l e s a r e i n t a c t and engaged i n a normal p e r i o d i c b r e a t h i n g p a t t e r n , l u n g volume d e c r e a s e s s l i g h t l y d u r i n g the b r e a t h h o l d p e r i o d ( L e f a n t e_t a l _ . , 1970). T h i s d e c r e a s e i n l u n g volume i s due to a de c r e a s e i n the r e s p i r a t o r y exchange r a t i o 82 FIGURE 18. SAR Discharge vs Lung Volume i n a H y p o t h e t i c a l 500  gram T u r t l e P o i n t (A) d i s c h a r g e r a t e a t 60 minutes of r e s t i n g lung volume (from f i g u r e 5, c o n t r o l p r o t o c o l ) P o i n t (B) d i s c h a r g e r a t e a t 60 minutes at e l e v a t e d lung volume (from f i g u r e 5, t e s t p r o t o c o l ) P o i n t (C) d i s c h a r g e r a t e a t 60 minutes of a b r e a t h h o l d with a 10 ml decrease i n lung volume 82a LUNG VOLUME (mis) 8 3 ( R E ) w h i c h i s r e s p r e s e n t e d a s V C 0 2 / V 0 2 w h e r e V i s t h e v o l u m e o f g a s m o v e d o v e r t i m e ( G l a s s e t _ a _ . , 1 9 8 3 ) . I n o t h e r w o r d s , t h r o u g h o u t t h e d u r a t i o n o f t h e b r e a t h h o l d , m o r e o x y g e n d i f f u s e s o u t o f t h e l u n g s t h a n C 0 2 d i f f u s e s i n t o t h e l u n g s . A c k e r m a n a n d W h i t e ( 1 9 7 9 ) h a v e m e a s u r e d b o t h t h e RE v a l u e s o f t u r t l e s o v e r e x t e n d e d p e r i o d s o f b r e a t h h o l d i n g a s w e l l a s t h e d e c r e a s e i n l u n g v o l u m e a s s o c i a t e d w i t h t h i s b r e a t h h o l d p e r i o d . F r o m t h e d a t a o f A c k e r m a n a n d W h i t e ( 1 9 7 9 ) , I h a v e e s t i m a t e d t h a t o v e r a 1 h o u r p e r i o d , l u n g v o l u m e i n t h e h y p o t h e t i c a l t u r t l e w i l l b e r e d u c e d b y a p p r o x i m a t e l y 10 m i s . T h e r e f o r e , t h e t u r t l e w i l l h a v e a l u n g v o l u m e o f 9 2 m i s a f t e r 1 h o u r o f b r e a t h h o l d i n g f r o m a s t a r t i n g v o l u m e o f 1 0 2 m i s . T h i s i s i l l u s t r a t e d a s p o i n t C i n f i g u r e 1 8 . T h i s v o l u m e c h a n g e r e s u l t s i n a d i s c h a r g e r a t e t h a t i s 4 2 % o f t h e p e a k v a l u e . I t m u s t b e n o t e d h o w e v e r , t h a t t h e e s t i m a t e d v a l u e s o f c h a n g e s i n r e c e p t o r d i s c h a r g e a s s o c i a t e d w i t h c h a n g e s i n l u n g v o l u m e h a v e b e e n e x t r a p o l a t e d f r o m a u n i l a t e r a l l y v a g o t o m i z e d t u r t l e . I n a n i n t a c t a n i m a l , t h e s e v a l u e s may b e s o m e w h a t d i f f e r e n t . T h i s e x e r c i s e d e m o n s t r a t e s t h a t t h e d i s c h a r g e v a l u e s we m e a s u r e d a t t h e e n d o f 1 h o u r o f m a i n t a i n e d l u n g i n f l a t i o n ( 4 4 % ) w e r e n8t a p p r e c i a b l y d i f f e r e n t f r o m t h o s e c a l c u l a t e d f o r a 1 h o u r b r e a t h h o l d p e r i o d i n a n o r m a l ( i n t a c t ) t u r t l e ( 4 2 % ) . T h e s e r e s u l t s h e l p v a l i d a t e t h e e x p e r i m e n t a l p r o t o c o l a s a p h y s i o l o g i c a l l y r e l e v a n t m e a n s o f e x a m i n i n g t h e a d a p t a t i o n r e s p o n s e o f t u r t l e s l o w l y a d a p t i n g p u l m o n a r y s t r e t c h r e c e p t o r s t o m a i n t a i n e d l u n g i n f l a t i o n s . 84 NOTE ON RAPIDLY ADAPTING PULMONARY STRETCH RECEPTORS As demonstrated i n f i g u r e 15, r a p i d l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s respond t o i n c r e a s e s i n l u n g volume w i t h an i n c r e a s e i n d i s c h a r g e . These r e c e p t o r s a re e a s i l y s t i m u l a t e d by n o x i o u s i n h a l e n t s and t h e i r r e f l e x i s m a n i f e s t i n the cough response ( P a i n t a l , 1977). S i x r a p i d l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s were r e c o r d e d from t h r o u g h o u t the s t u d y , but no d e t a i l e d a n a l y s i s was conducted on t h i s r e c e p t o r group. RELATIVE ROLES OF TONIC AND PHASIC DISCHARGE IN. THE CONTROL OF BREATHING IN TURTLES AND GENERAL CONCLUSIONS The c o n t r o l o f b r e a t h i n g i s a c h i e v e d by c e n t r a l i n t e g r a t i o n o f i n f o r m a t i o n a r i s i n g from chemoreceptors ( b o t h c e n t r a l and p e r i p h e r a l ) , r e s p i r a t o r y muscle a f f e r e n t s and pulmonary r e c e p t o r s . G e n e r a l l y , the c e n t r a l and p e r i p h e r a l chemoreceptors go v e r n the o v e r a l l l e v e l o f v e n t i l a t i o n w h i l e the s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s i n t e r a c t w i t h the c e n t r a l r e s p i r a t o r y rhythm g e n e r a t o r t o produce a c h a r a c t e r i s t i c b r e a t h i n g p a t t e r n . Pulmonary s t r e t c h r e c e p t o r s were c l a s s s i c a l l y i n v o k e d i n the H e r i n g - B r e u e r i n s p i r a t o r y o f f - s w i t c h r e f l e x . P r e l i m i n a r y e x a m i n a t i o n s i n t o the f u n c t i o n a l c h a r a c t e r i s t i c s o f s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s have l e d to the c o n c l u s i o n t h a t the v a g a l l y - m e d i a t e d i n h i b i t o r y i n f o r m a t i o n a r i s i n g from the SARs i n t e r a c t s w i t h the c e n t r a l r e s p i r a t o r y rhythm g e n e r a t o r to 85 terminate the i n s p i r a t o r y phase of the b r e a t h i n g c y c l e ( E u l e r et a l . . 1970; C l a r k and E u l e r , 1972). C l a r k and E u l e r (1972) p o s t u l a t e d t h a t the p h a s i c , r a t h e r than the t o n i c component of SAR d i s c h a r g e was of primary importance i n the c o n t r o l of the b r e a t h i n g c y c l e . More r e c e n t l y however, s t u d i e s have demonstrated t h a t not only i s t o n i c i n f o r m a t i o n important, p a r t i c u l a r l y i n r e g u l a t i n g TE ( G r u n s t e i n et. al. . , 1975), but t h a t e x p i r a t o r y d u r a t i o n and i n s p i r a t o r y d u r a t i o n may be r e g u l a t e d through separate r e f l e x mechanisms (Agostoni e_t §1.., 1985). There i s no doubt t h a t v a g a l l y - m e d i a t e d i n f o r m a t i o n a r i s i n g from SARs does p l a y a major r o l e i n producing and m a i n t a i n i n g the b r e a t h i n g p a t t e r n (Bradley, 1977). The c o n t r o v e r s y however l i e s i n the r e l a t i v e importance of the t o n i c and p h a s i c components of SAR d i s c h a r g e i n the c o n t r o l of the b r e a t h i n g p a t t e r n . R e s u l t s presented by G r u n s t e i n e_t a l , ( 1975) i n d i c a t e that the t o n i c component of SAR d i s c h a r g e adapts completely to maintained i n c r e a s e s i n lung volume. Other s t u d i e s however, have demonstrated t h a t there i s o n l y a p a r t i a l a d a p t a t i o n of the t o n i c component of SAR d i s c h a r g e (D'Angelo and Agostoni, 1975; Davenport et. al.. , 1981; Muza and F r a z i e r , 1983). T u r t l e s o f t e n undergo c h r o n i c changes i n f u n c t i o n a l r e s i d u a l c a p a c i t y to combat changes i n body weight i n an attempt to m a i n t a i n n e u t r a l buoyancy. In doing so, there i s a dramatic and p e r s i s t a n t change i n the b r e a t h i n g p a t t e r n of these animals (Milsom and Chan, 1986). I f the t o n i c (volume-related) component of SAR d i s c h a r g e completely adapts to changes i n FRC as suggested 86 by G r u n s t e i n §__,_.• (1975), t h e n the p e r s i s t a n t changes i n b r e a t h i n g p a t t e r n o b s e r v e d by Milsom and Chan (1986) must be the r e s u l t o f changes i n the c e n t r a l r e s p i r a t o r y rhythm g e n e r a t o r . I f on the o t h e r hand, SARs do not c o m p l e t e l y adapt to changes i n FRC as suggested by D'Angelo and A g o s t o n i (1975), Davenport e_t a l . (1981) and Muza and F r a z i e r (1983), then one c o u l d p o s t u l a t e t h a t the t o n i c ( v o l u m e - r e l a t e d ) component o f SAR d i s c h a r g e i s i m p o r t a n t i n p r o d u c i n g and m a i n t a i n i n g the b r e a t h i n g p a t t e r n changes o b s e r v e d i n t u r t l e s e x p e r i e n c i n g a l t e r a t i o n s i n FRC. T h i s s t u d y has demonstrated t h a t n e i t h e r the low t h r e s h o l d nor the h i g h t h r e s h o l d s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s adapt to l o n g term (1 hour) m a i n t a i n e d l u n g i n f l a t i o n s . At the end o f 1 hour o f m a i n t a i n e d e l e v a t e d l u n g volume, the low and h i g h t h r e s h o l d SARs have o n l y adapted 80% and 30% from t h e i r peak d i s c h a r g e r a t e s r e s p e c t i v e l y . F u r t h e r m o r e , l o n g term l u n g i n f l a t i o n w i t h a i r produced o n l y minor ( a l b i e t s t a t i s t i c a l l y s i g n i f i c a n t ) d e c r e a s e s i n low t h r e s h o l d SAR dynmaic i n f l a t i o n r e s p o n s e s compared to those r e c o r d e d p r i o r to the p e r i o d o f m a i n t a i n e d i n f l a t i o n . The dynamic i n f l a t i o n r e s p o n s e s o f the h i g h t h r e s h o l d SARs were unchanged by 1 hour o f m a i n t a i n e d l u n g i n f l a t i o n w i t h a i r . I n c o n c l u s i o n , s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s do r e t a i n s i g n i f i c a n t t o n i c and p h a s i c d i s c h a r g e w i t h p r o l o n g e d l u n g i n f l a t i o n . C o n s e q u e n t l y , the v o l u m e - r e l a t e d ( t o n i c ) component o f SAR d i s c h a r g e i s l i k e l y to be i n s t r u m e n t a l i n p r o d u c i n g and m a i n t a i n i n g the changes i n b r e a t h i n g p a t t e r n t h a t are a s s o c i a t e d 87 w i t h c h r o n i c a l t e r a t i o n s i n l u n g volume i n t h e s e a n i m a l s . R e s u l t s from t h i s s t u d y tend to s u p p o r t those o f D'Angelo and A g o s t o n i (1975), Davenport §_t al_. , (1981) and Muza and F r a z i e r (1983). I t must be noted t h a t I do not a t t r i b u t e these changes i n b r e a t h i n g p a t t e r n s o l e l y to a f f e r e n t i n p u t a r i s i n g from s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s . The o v e r a l l b r e a t h i n g p a t t e r n o f t h e s e a n i m a l s i s l i k e l y to be produced by the i n t e g r a t i o n o f i n f o r m a t i o n from c h e m o r e c e p t o r s , s l o w l y a d a p t i n g pulmonary s t r e t c h r e c e p t o r s and the c e n t r a l r e s p i r a t o r y i n t e g r a t i v e c e n t e r s . 88 LITERATURE CITED Ackerman, R.A. and F.N. White. 1979. C y c l i c Carbon Dioxide Exchange i n the T u r t l e , Pseudemvs s c r i p t a . P h y s i o l . Zool. 52: 378-389. Adrian, E.D. 19 3 3. A f f e r e n t Impulses i n the Vagus and Their E f f e c t on R e s p i r a t i o n . J. P h y s i o l . 79: 332-358. Agostoni, E., G. C i t t e r i o and S. P i c c o l i . 1985. 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