RESPIRATORY RESPONSES TO ADRENOCORTICOTROPIN AND DEXAMETHASONE IN UNANESTHETIZED GOATS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY BARRY ERNEST WATKINS 1971 ’ I LIBRARY Michigan Stab Univcxsity : mane av 1’ IIIIAE & SIIIIS' ABSTRACT RESPIRATORY RESPONSES TO ADRENOCORTICOTROPIN AND DEXAMETHASONE IN UNANESTHETIZED GOATS BY Barry Ernest Watkins Adrenocortical influences on chemically regulated respiratory excitability were measured subsequent to intra— muscular administration of adrenocorticotrOpin (80 I.U.) and dexamethasone (2.5 mg) to adult female goats with hormone treatment schedules of 12 and 36 hours. Hormone injections were given at 12 and 3 hours before carbon dioxide (C02) inhalation in the 12 hour regimes, while the 36 hour treat- ments involved similar hormone injections at 36, 24, 12 and 3 hours before the inspiratory studies. Minute ventilation, respiratory frequency, fraction of expired oxygen, fraction of expired C02, rectal temperature, heart rate, mean arte- rial blood pressure, arterial pH, arterial bicarbonate con- centration and arterial CO tension were monitored during 2 the administration of inspiratory gases consisting of room air, and gas mixtures of 2%% CO2 plus 21% O2 in nitrogen and 5% CO2 plus 21% O2 in nitrogen. These measurements allowed the calculation of tidal volume (VT), respiratory dead space, alveolar ventilation (VA) and oxygen consumption. Estima- tions of cisternal cerebrospinal fluid (CSF) bicarbonate Barry Ernest Watkins concentration, CSF CO2 tension and CSF pH were made from corresponding arterial values by employing comparisons of arterial and CSF measurements existent in the literature. No significant changes occurred in respiratory frequency, blood pressure, heart rate, rectal temperature, arterial bicarbonate concentration or CSF bicarbonate con- centration in any of the groups following the development of hypercapnic respiratory drive. Oxygen consumption was not significantly affected by any of the hormone treatments. Additionally, mean rectal temperatures of the treated groups were consistently less than those of control. Mean arterial blood pressure during both dexametha- sone treatments was 17 mm Hg greater than those values of either control or of the two adrenocorticotrOpin (ACTH) treatments. This effect was presumably associated with high-level glucocorticoid potentiation of vasoconstrictor activity. Further, the bicarbonate concentration in both the blood and CSF compartments was significantly elevated in the 36 hour ACTH regime over that found in any other group, probably resulting from the develOpment of metabolic alka- losis due to the enhanced mineralocorticoid activity induced by this treatment. Tidal volume, VA’ tration and CSF H+ concentration all were elevated upon arterial hydrogen ion (H+) concen- increasing inspired CO2 content. The increased respiratory activity during induced hypercapnia was related to the Barry Ernest Watkins elevated CSF H+ concentration which presumably stimulated the central chemoreceptors. Correlations between HA and blood or CSF CO2 tension (PC02) indicated that all treatment groups except the 36 hour ACTH regime exhibited greater respiratory excitability than control animalsysince the NA of the hormonally treated animals was greater than that of control animals at all measured CSF or arterial PC02° Evaluation of YA in terms of arterial pH indicated that all hormone treatments other than the 12 hour dexamethasone group exhibited greater respira- tory excitability than control animals throughout the mea— sured range of arterial pH. Comparison of HA as a function of CSF H+ concentration showed that all hormone treatments consistently increased respiratory excitability above con- trol by depressing the ventilatory response set-point for CSF H+ concentration by approximately 3.0 nM H+/kg H20. The ventilatory response sensitivity was essentially unaltered since the magnitude of change in TA for unit change in H+ concentration was similar in all treatment regimes to that of control. Since both the ACTH and dexamethasone treatments induced nearly identical increases in respiratory activity at specific levels of CSF pH, the effective mediators of these response changes were presumably glucocorticoids. The ability of ACTH to alter ventilation was thus associated with this hormone's action to enhance adrenocortical hormone release. The similarity of the 12 and 36 hour responses Barry Ernest Watkins suggests that the glucocorticoid effects on respiratory mechanisms occur within 12 hours after initiation of hormone treatment. It is possible that the site of glucocorticoid action is directly on the medullary respiratory control center to decrease its set-point for respiratory response to CSF H+ concentration. The glucocorticoid effect might also be to enhance chemoreceptor activity at either central or peripheral sites. Finally, glucocorticoids may act to alter the strength of an input to the respiratory control center from some source other than that of the chemore- ceptors. RESPIRATORY RESPONSES TO ADRENOCORTICOTROPIN AND DEXAMETHASONE IN UNANESTHETIZED GOATS BY Barry Ernest Watkins A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1971 6 I ACKNOWLEDGMENTS I am sincerely grateful to Dr. Gail D. Riegle, my major advisor, for originally providing me with a "home" within the department. I am equally appreciative of my major advisor's patient counsel and his readily available assistance throughout the course of my Master's program. I wish also to thank Dr. W. R. Dukelow, of my guidance committee, for providing advise and support at most fitting occasions. The friendship and helpfulness of everyone on the Endocrine Research Unit staff has been a constant source of encouragement to me. Recognition is given to Dr. S. R. Heisey for his aid in the selection of an apprOpriate experimental design for this study. I also acknowledge the other members of my advisory committee, Dr. S. R. Heisey and Dr. E. P. Reineke for the role that they played in regards to the furtherance of my vocational goals. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . l LITE MT URE REVIEW 0 O O O O O O O O O O O O O O O O O 2 Respiratory Control . . . . . . . . . . . . . . . 2 Characterization and Effects of Adreno- corticotrOpin . . . . . . . . . . . . . . . . . lO Glucocorticoid Actions . . . . . . . . . . . . . 15 Adrenocortical Activation Following Respiratory Stress . . . . . . . . . . . . . l9 Hormonal Effects on Respiratory Excitability . . 21 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 24 Atlimals O O O O O O O O O O O O O O O O O O O O O 24 Experimental Conditions . . . . . . . . . . . . . 24 Experimental Design . . . . . . . . . . . . . . . 28 Measured Variables . . . . . . . . . . . . . . . 31 Calculated Parameters . . . . . . . . . . . . . . 34 Statistical Analysis . . . . . . . . . . . . . . 37 RESULTS 0 O O O O O O O O O O O O O O O O O O 0 O O O 38 Response Changes Due to C02 Inhalation . . . . . 38 Response Differences Between Hormone Treatment Groups . . . . . . . . . . . . . . . 39 Blood Pressure, Heart Rate, Rectal Temperature and Oxygen Consumption . . . . 39 ReSpiratory Frequency, Tidal Vblume and Alveolar ventilation . . . . . . . . . 40 Arterial PC02: pH and Bicarbonate concentratlon O O O O O O O O O O O O O O O 40 Cerebrospinal Fluid Pcoz, pH and Bicarbonate . . . . . . . . . . . . . . . . 42 Relationships Between Alveolar ventilation and Blood and CSF Chemistry . . . . . . . . 43 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 57 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 70 REFEENE LIST 0 O O O O O O O O O O O O O O O O O O 73 iii Table 1. LIST OF TABLES Page Effects of hormone treatments and inspiratory C02 on arterial blood pressure, heart rate and rectal temperature . . . . . . . . . . . . 46 Effects of hormone treatments and inspiratory C02 on respiratory frequency, tidal volume, and alveolar ventilation . . . . . . . . . . . 47 Effects of hormone treatments and inspiratory C02 on arterial PC02: arterial bicarbonate, and arterial pH . . . . . . . . . . . . . . . 48 Effects of hormone treatments and inspiratory C02 on CSF PC02r CSF bicarbonate, and CSF pH 0 O O O O O O O O O O O O O O O I O O O O O 49 Effects of hormone treatments on oxygen consumption 0 O O I O O O O I O O O O I O O O 50 iv Figure 1. LIST OF FIGURES Environmental chamber for data collection Treatment schedules . . . . . . Effect of hormone treatments on ventilatory response to induced of arterial PCO2 . . . . . . . Effect of hormone treatments on ventilatory response to induced in arterial pH . . . . . . . . Effect of hormone treatments on changes the changes the ventilatory response to estimated changes of CSF H+ concentration Page 27 29 52 54 56 INTRODUCTION The first reports concerning the effects of stages of the menstrual cycle and pregnancy on ventilation appeared in the literature nearly 60 years ago. Subsequent investi- gations suggested that several steroid hormones may be responsible for increasing respiratory excitability. These studies, conducted with untrained human subjects under poorly controlled controlled conditions, used the partial pressure of either alveolar or arterial carbon dioxide as an index of the level of reSpiratory activation. The quan- titative effect of any hormone on respiration has yet to be determined. The site of hormonal influence and the mecha- nism of action have likewise not yet been elucidated. The present study was designed to attempt a more extensive and precise evaluation of adrenocortical influence on respira- tory excitability. LITERATURE REVIEW There is evidence that certain gonadal (Hasselbalch and Gammeltoft, 1915; Loeschcke and Sommer, 1944; Heerhaber et al., 1948; Goodland et al., 1953; Huang and Lyons, 1966; Lyons and Huang, 1968) and adrenal steroids (Koepchen, 1953) increase respiratory activity. Koepchen et a1. (1954) suggested that ACTH may have a similar stimulatory effect upon respiration. In order to adequately evaluate mechanisms of hormonal action on respiration, it is necessary to present a brief review of respiratory control. Consideration must also be given to the multiple physiological effects of those hormonal agents employed in the course of this research. Respiratory,Control Gross localization of the respiratory control center was first performed by Julian Legallois in 1811 (Lambertson, 1968). By progressively ablating increasingly primitive areas of rabbit brain, he isolated the site of the control center to within the medulla. He found that the medulla alone was capable of generating spontaneous rhythmical respiration. This function of the medulla has since been well substantiated (Hoff and Breckenridge, 1948; Brodie and Borison, 1957; Wang et al., 1957). Lumsden (1923 and 1924) was the first investigator to describe the neural interactions by which the medullary center induces and regulates rhythmic breathing. He showed that extramedullary influences from the vagus and pontile pneumotaxic centers make possible the medullary rhythm. Lumsden found that when vagal and pontile-medullary connec- tions were severed, respiratory cyclic activity stOpped in the state of an inspiratory cramp. Lumsden's findings led to further analysis of the genesis of rhythmicity within the medulla. By observing the effects of stereotaxic stimulation of the internal medulla, several investigators (Pitts et al., 1939; Brookhart, 1940; Ngai and Wang, 1957; Cohen and Wang, 1958) were able to differentiate inspiratory and expiratory groups of neurons. Pitts et a1. (1939) found a spatial separation of these groups, with inspiratory neurons generally lying dorsal and caudal to the expiratory cell groups. On the other hand, Brookhart (1940) reported the groups to be diffuse and inter- mingled with one another, rather than existing as separate and discrete neurological centers. With the discovery of these intramedullary cell groups, it became evident that understanding of respiratory control was dependent on knowledge of the basic interactions of the two cell groups which constitute the respiratory control center. Upon direct stimulation of the inspiratory cell group (Haber et al., 1957), the inspiratory neurons in the control center were activated via cellular recruitment as evidenced by increased inSpiration amplitude and the associated increase in impulse frequency occurring in the phrenic nerve. Any input causing an increased activity level in either the inSpiratory or expiratory cell groups also brings about a simultaneous inhibitory influence upon the Opposed group (Salmoiraghi and Burns, 1960). The inspiratory group is believed to display spontaneous self re-excitation in initiating the inspiratory phase of the respiratory cycle (Burns, 1963). Cohen and Wang (1959) showed that two types of "phase spanning" neurons (inspiratory-expiratory and expiratory—inspiratory neurons) in the medulla and lower pontile region provide a modulating influence upon the medullary control center. The inspiratory-expiratory neurons start firing during inspiration, reaching a peak activity level at the end of inspiration, and then stOp firing during expiration. It was believed that these neurons inhibit inspiration. Conversely, neurons of the expiratory-inspiratory type are active from the middle of expiration until mid-inspiration, presumably acting to inhibit expiration. There is evidence for further modulation of the medullary respiratory control center by certain areas of the central nervous system. According to Lumsden (1923 and 1924), the pontile pneumotaxic center supplies constant inhibition to the inspiratory cell group. Fink et al. (1962) postulated an excitatory influence on respiration originating from an area between the superior and mid- collicular level in cats because decerebration rostral to the superior colliculus increased minute ventilation and respiratory frequency more than did mid-collicular decere- bration. A major afferent to the respiratory control center is from central chemoreceptors located either on the surface of the medulla (Mitchell et al., 1963) or at an internal medullary site (Pappenheimer et al., 1965; Fencl et al., 1966). The regulatory activity of the respiratory control center is also modified by inputs from the periphery. In 1889, Head found that decreasing lung volume provided a stimulus for inspiration that was mediated through the vagus. This was a reversed Hering-Breuer response, where lung deflation apparently decreased the inhibitory input from the pulmonary stretch receptors. Chemoreceptive tissue in the carotid and aortic bodies has also been identified (Comroe and Schmidt, 1938; Comroe, 1939) as having reflex influences upon respiration. The most comprehensively studied peripheral chemo- receptor, the carotid body, has a large blood flow, allowing it to rapidly and reliably respond to blood chemistry alter- ations in the arterial supply which perfuses it (Daly et al., 1954). A significant arterio-venous oxygen difference across the carotid bodies of the cat subsequent to partial common carotid occulusion suggested that the receptors have appreciable metabolic rates (Daly et al., 1954). Under normal conditions, tonic afferent activity can be monitored in the sinus nerve, which originates from the carotid body (Eyzaguirre and Lewin, 1961). Perfusing carotid or aortic bodies with fluids containing high C02 tensions or low partial pressures of arterial oxygen (Comroe and Schmidt, 1938; Comroe, 1939) results in a stimulation of respiration. Decreasing the pH of similar perfusates was shown by Schmidt et a1. (1939) to enhance respiration. Decreasing blood flow through the carotid bodies induced respiratory stimulation, where rapid metabolic utilization of oxygen and formation of CO and other acid metabolites in the slowly moving blood 2 were postulated to be the ultimate source of stimulation to the carotid receptors (Daly et al., 1954). Hornbein and Roos (1963) reported that afferent carotid discharge fre- quency was a function of arterial pH rather than of arterial CO2 tension (PaC02)' The work of Gemmill and Reeves (1933) suggested that although peripheral chemoreceptors are important in monitoring changes of the partial pressure of oxygen, they are not necessary for a respiratory response to hypercapnia. More recent investigations by Dutton (1967) indicated that oscillations of the C02 tension of blood perfusing the carotid bodies results in greater increases in ventilation than do comparable steady state elevations of PaC02° This work suggests that PaC02 oscillations resulting from cyclic changes in the alveolar PC02 may be sensed in the carotid bodies and provide a fine adjustment of respiration. As early as 1926, Gesell and Hertzman observed that the cisternal cerebrospinal fluid (CSF) pH was different from that of arterial samples. The inhalation of CO2 rich gases results in increasing the hydrogen ion concentration in both arterial blood and in cerebrospinal fluid. Robin and Bromberg (1959) hypothesized a more rapidly achieved equilibrium of CO between blood and CSF than equilibriums 2 with either hydrogen or bicarbonate ions. Since the partial pressure of CO2 in internal jugular blood is essentially in equilibrium with CSF PCOZI Lambertson et a1. (1961) were of the opinion that the parameters of CSF hydrogen ion concen- tration and internal jugular venous PCO2 are equally valid indicators of ventilatory drive to central respiratory chemoreceptors in the steady state. Several groups of investigators (Leusen, 1954; Mitchell et al., 1963; Pappenheimer et al., 1965) have indicated that cerebral ventricular perfusion of bicarbonate buffer solutions containing high hydrogen ion concentrations or low bicarbonate concentrations acted as a stimulus to respiration. A depression of respiration was observed when CSF perfusates contained low hydrogen ion concentrations. These effects were mediated centrally since respiration was modified before changes were detected in arterial blood chemistry (Leusen, 1954). However, there remain conflict- ing vieWpoints concerning the exact location of the central chemoreceptor site. Direct application of acetylcholine, nicotine or acidic solutions (Mitchell et al., 1963) upon the ventro- lateral surface of the medulla caused a prompt and marked stimulation of respiration. These data support the view that the chemoreceptor site is on the medullary surface, and therefore anatomically separated from the respiratory control center. Other experimental techniques have provided results which are inconsistent with the hypothesis of superficial medullary chemoreceptors. Leusen (1954) used boric acid/ sodium borate buffers in solutions perfusing the brain ventricles, which allowed him to alter CSF hydrogen ion concentration independently of CO2 or of bicarbonate. He observed no ensueing changes in respiration following intra- ventricular perfusions containing high boric acid concentra- tions. It was found that dynamic respiratory responses to administration or withdrawal of inspired CO2 were more rapid than CSF changes, but appeared to lag behind variations of arterial pH, PaCOZ or alveolar PCO2 (Fuleihan et al., 1963; Lambertson et al., 1965). The fact that respiratory responses to inspired CO2 occur at a time intermediate between detectable CSF and Pa changes suggests that the chemoreceptor location is CO suchzthat it is simultaneously affected by chemical changes in both blood and CSF. Lambertson et a1. (1961) found that the total respiratory response to inhaled CO2 was decreased by 45% when the respiratory acidosis was compensated with bicarbonate infusion. They concluded that 45% of the reSpi- ratory response was induced by arterial changes following CO2 inhalation, leaving 55% of the response due to CSF changes. In that there is no appreciable blood-CSF gradient for C02 (Lambertson et al., 1961), Pappenheimer and his associates (1965) concerned themselves with bicarbonate and hydrogen ion gradients between CSF and blood. When the CSF was perfused with solutions of varying bicarbonate concen- trations, they found that alveolar ventilation in goats was a single function of the hydrogen ion concentration at a point approximately % the way down a bicarbonate concentra- tion gradient between the medullary-CSF surface and an idealized interface at the capillary-glial boundary. This point along the gradient was designated as the location of the central chemoreceptor, in that ventilation was a single function of the calculated hydrogen ion (H+) concentration at this site. According to this hypothesis, the ultimate regulatory mechanism adjusting ventilation to various acid- base disturbances is a bicarbonate pump which maintains simultaneous gradients of hydrogen and bicarbonate HCO3 ions between blood and CSF (Pappenheimer, 1966). 10 Fencl et al. (1966) showed that in the steady state, alveolar ventilation of goats is directly related to CSF hydrogen ion concentration, regardless of chronic metabolic acid-base disturbances. In these studies, no net ionic fluxes between the CSF and blood compartments were observed. Assuming a bicarbonate pump at the glial-capillary boundry adjusted ionic movement, it was concluded that the ionic concentrations in the CSF were essentially identical with those of brain interstitial fluid. The work of Fencl's group thus suggested that under steady state conditions, measurements of CSF H+ concentration is a valid reflection of hydrogen ion concentration at a brain interstitial chemoreceptor site. Characterization and Effects of Adrenocorticotropin Hypophysectomy results in marked atrophy of the adrenal cortex (Smith, 1930) because the pituitary releases a hormone, adrenocorticotropin (ACTH), capable of maintain- ing adrenocortical development. AdrenocorticotrOpin is a randomly coiled globular protein consisting of thirty-nine amino acid residues with a total molecular weight of about 4,500. The complete amino acid sequence of this protein has been determined for several mammals including the sheep, pig and cow (Li et al., 1958; Harris, 1960). There is minimal interspecies variation in the amino acid sequence. The COOH- terminal is phenylalanine and N-terminal is serine, which 11 is essential for characteristic action of the hormone (Sheperd et al., 1956). Most variation among species occurs at positions between residues 25 and 33 of the polypeptide chain (Li et al., 1958). Adrenocorticotropin is synthesized and stored in the adenohypophysis. This area of the pituitary is highly vascular, receiving its blood supply from the hypothalamic- hypophyseal portal system (Wislocke, 1937). The portal system provides the means by which the hypothalamus controls ACTH secretion (Harris, 1948). Cells in the median eminence of the hypothalamus are rich in small polypeptides which activate synthesis and release of ACTH (vernikos-Danellis, 1965), and thus cause adrenal cortical activation (Matsuda et al., 1964). According to Schally et a1. (1958), the corticotropin releasing factor (CRF) peptide is similar in amino acid composition and sequence to that of vasopressin. This releasing factor is produced by cells located in the hypothalamic median eminence and secreted into the hypo- physeal portal system, with CRF acting as the hormonal messenger to target cells within the adenohypophysis (Guillemin, 1964). Hypophyseal release of ACTH is thought to be con- trolled by at least two negative feedback mechanisms. Several investigators (McCann et al., 1958; Smelik and Sawyer, 1962; Chowers et al., 1963; Davidson and Feldman, 1963) have shown that adrenocortical hormones injected or 12 implanted into the rat's hypothalamus bring about a decreased level of adrenal cortical function, and cause pronounced adrenocortical atrophy during chronic adminis- tration. In 1964, Beaven et al. found that intravenous injections of 10 mg of dexamethasone, a synthetic gluco- corticoid, completely suppresses adrenocortical secretion in sheep for twelve hours. This inhibition is of central origin since the adrenals were still responsive to exogenous ACTH. Further, Brodish and Long (1962) reported that plasma CRF decreased after cortisol treatment of hyp0physectomized rats. These results support the concept that adrenal ste— roids provide negative feedback by inhibiting the hypotha- lamic release of CRF. When cortisol was injected directly into the pituitary (Smelik and Sawyer, 1962; Chowers et al., 1963), no decrease in adrenocortical activity ensued. It was concluded that cortisol does not directly inhibit the pituitary. However, data in support of steroid feedback on the pituitary was presented by Russel et a1. (1969), who noted that dexamethasone injected into the adenohypophysis was able to prevent ACTH release in response to eleVated CRF release. The findings of Kitay et al. (1959) suggested that ACTH has the capability of inhibiting its own release, although was uncertain whether this action occurs at the hypothalamus or hypophysis. Brodish and Long (1962) ob- served that the CRF concentration in the peripheral blood of rats increased after hypophysectomy and adrenalectomy, l3 probably resulting from loss of inhibition of ACTH upon the hypothalamic synthesis or release of CRF. Other extra-adrenal effects of ACTH have been recognized. Adrenocorticotropin is known to possess some melanocyte-stimulating hormone (MSH) activity which is attributable to similarities between MSH and the N-terminal ACTH amino acid sequences (Li et al., 1960). Hollenberg et a1. (1961) hypothesized that the acknowledged lipolytic activity of ACTH on adipose tissue is brought about by activation of a lipase other than lipoprotein lipase to induce enzymatic hydrolysis of triglycerides. Adrenocor— ticotropin is also known to have an anabolic effect upon protein which is dependent on the maintenance of permissive levels of glucocorticoids (Engel and Feredicks, 1959). Torda and Wolff (1952) studied the effects of ACTH on the electrical activity of the brain. They made use of the electroencephalogram and the sensitivity to a convulsion- inducing drug as indices of brain excitability in adrenal- ectomized and hypophysectomized rats. Brain activity increased at from one to three minutes after ACTH injections, while chronic ACTH treatment which was of three to four days duration resulted in decreasing electrical activity of the brain. The most documented effect of ACTH is its action upon the adrenal cortex. Removing the ACTH influence by hypOphysectomy results in atrophy of the fasciculata and reticularis cell layers of the adrenal cortex (Deane and l4 and Greep, 1946). Conversely, ACTH injections result in hypertrOphy of these same layers (O'Donnell et al., 1951). The ACTH induced adrenal hypertrOphy is in part due to incorporation of amino acids into adrenal proteins (Farese, 1965). Stachenko and Giroud (1959) reported that ACTH added to slices of bovine fasciculata and reticularis cell cul- tures causes an increase of the corticosteroid concentration in the medium. The glomerulosa layer of the adrenal cortex, whose histological integrity is essentially independent of ACTH, is responsible for the synthesis of aldosterone. The effect of ACTH on aldosterone release is not as clear as ACTH effects on other portions of the adrenal cortex. Data of Bartter et a1. (1959) suggest that ACTH does stimulate aldosterone release in man, whereas Axelrad (1954) found no increase of plasma aldosterone after ACTH treatment. Stachenko and Giroud (1959) showed that ACTH added to glomerulosa cell cultures does not significantly change the production rate of either glucocorticoids or aldosterone. The effects of ACTH on the zona glomerulosa are thought to be greatly overshadowed by the more specific effects of the renin-angiotensin system to stimulate aldosterone release (Tobian, 1960). The primary effect of ACTH on the adrenals is to stimulate the synthesis and release of adrenocortical hormones which are high in glucocorticoid activity. Recent investigations (Saffran and Rowell, 1969; Jaanus et al., 15 1970) have utilized in vitro adrenal gland perfusion. By continuously monitoring corticosterone output it was found that, after a five to ten minute lag period, corticosterone output varies directly with the ACTH concentration and the duration of its perfusion. The response to a constant ACTH infusion decreases with time, suggesting an adaptation phenomenon. The perfusion of isolated adrenal cells, of which 90% are of the reticularis or fasciculata type, pro- vides another method to assay the effect of ACTH on gluco- corticoid release (Kitabchi and Sharma, 1971). Results from this perfusion assay indicated that both ACTH and 3', S'-cyc1ic adenosine monophosphate nucleotides stimulate corticosterone production to an equal extent, implying that the response to ACTH may be mediated through this nucleotide. Glucocorticoid Actions The large volume of information that has accumulated concerning glucocorticoid effects has been extensively reviewed (Moon, 1961; Eisenstein, 1967). Circulating gluco- corticoids alter carbohydrate metabolism by stimulating hepatic glycogen synthesis (Glenn, 1961). The hyperglycemia following glucocorticoid administration indicates that these steroidal hormones may act to decrease peripheral glucose utilization (Jeanrenaud and Renold, 1960). There is evi- dence suggesting that glucocorticoids exhibit insulin antagonistic properties on fat metabolism, causing an inhibition of hepatic lipogenesis while enhancing lipolysis 16 in adipose tissue (Brady et al., 1951; Fain, 1964). Gluco- corticoids apparently act to increase whole body protein catabolism as evidenced by elevated urinary nitrogen excre- tion (Long et al., 1940), while inducing increased hepatic amino acid accumulation for protein synthesis (Weber et al., 1965). Cortisone has been reported to antagonize the vita- min D dependent uptake of calcium from the small intestine (Harrison and Harrison, 1960). Large, non-physiological doses of glucocorticoids have also been demonstrated to alter immunological and inflammatory reactions, and to inhibit wound healing (ijrnboe et al., 1951; Holden and Adams, 1957; Weissmann and Thomas, 1964). Natural glucocorticoids exhibit some mineralocorti- coid activity, and it has been shown that significant mineralocorticoid effects result from exposure to substan- tial levels of glucocorticoids. Administration of aldo- sterone and deoxycorticosterone acetate causes decreased urinary sodium and increased urinary potassium excretion (Ross et al., 1959; Ferrebee et al., 1941; Ganong and Mulrow, 1958). Conn's (1955) description of primary aldo- steronism includes the development of metabolic alkalosis and decreased renal tubular reabsorption of water. Bartter and Fourman (1962) reported that after injection of ACTH in humans, potassium and hydrogen ion excretion increased independently of changes in sodium excretion. l7 Massive treatment doses of cortisol have been reported to increase cardiac output and decrease peripheral resistance without significantly altering arterial blood pressure (Sambhi, 1965). The findings of Zweifach (1960) indicate that glucocorticoids function to potentiate cardio- vascular responses to catacholamine vasoconstrictors. On the other hand, Small et a1. (1959) were not able to demon— strate the existence of such potentiation in the pulmonary arterioles of Starling heart-lung preparations. Adrenocortial steroids provide negative feedback upon the hypothalamic CRF releasing cells (Beaven et al., 1964; Russel et al., 1969), suggesting that specific recep- tor sites for glucocorticoids exist in the central nervous system. The brain is known to take up labeled blood cortisol, which disappears more rapidly from blood plasma than from the brain, suggesting that the steroid is accumu- lated in the brain (Peterson and Chaikoff, 1963; Walker et al., 1971). Touchstone et a1. (1966) demonstrated that whole human brain homogenate contains a cortisol concentra- tion of 400 ug/lOO gm compared to 15 ug/lOO ml in blood. McEwen et a1. (1969) postulated that the rat brain has two separate mechanisms for the uptake of labeled corticosterone: (1) a generalized concentrating mechanism in the whole brain; and (2) a limited active uptake in specific areas of the septum and hippocampus. 18 Electro-shock seizure threshold has been used as a measure of brain excitability. Woodbury and Sayers (1950) demonstrated that cortisone treatment caused a decrease in the shock threshold, indicating an increase in whole brain electrical activity. On the other hand, analysis of electro- encephalograms after cortisone or ACTH administration to medical students showed no change in electrical activity (Friedman and Engel, 1956). It is possible to monitor electrical activity at various isolated points within specific brain structures by the use of multiple or single unit recording electrodes. Feldman et a1. (1961) made unit recordings of cat brain stem potentials evoked by sciatic nerve stimulation. Increases in amplitude of the evoked responses in hypothalamic and midbrain reticular sites were noticed within five to fifteen minutes after cortisol injection. Similar potential changes occurred in these same areas two hours after ACTH treatment. Feldman et a1. concluded that the induced response was directly due to the steroid hormone, and that the increased potential amplitude indicated an alteration of membrane potential to lower the threshold for synaptic transmission. Intravenous injection of cortisol also acts to alter the spontaneous rate of firing of hypothalamic and midbrain reticular units in cats (Slusher et al., 1966). After injecting cortisone into the third cerebral ventrical, rapid increases in discharge frequency occurred in units of the lateral hypothalamus and of the hippocampus. However this l9 procedure did not indicate noticeable changes in midbrain reticular unit activity following such cortisone injections (Feldman, 1966). The possibility exists that glucocorticoids may only indirectly effect brain activity by functioning to alter the availability of certain chemical transmitter substances. Steiner et a1. (1969) have demonstrated that acetylcholine and dexamethasone have Opposite effects upon the activity of mesencephalic units in the rat. Maas and Mednieks (1970) showed that hydrocortisone, added to cultures of rat cere- bral cortex, produces increased norepinepherine uptake. Adrenocortical Activation Following Respiratory Stress Certain relationships between respiration and adreno- cortical function have been formulated. One route of inves- tigation has been to elucidate the influence of changes in respiratory drive and alterations of arterial acid-base chemistry upon adrenal release of l7-hydroxycorticosteroids. It has been reported that moderate hypoxia (100 mm Hg in- spired oxygen) results in increased peripheral plasma 17- hydroxycorticosteroid concentration in man (Hale et al., 1957). However, Biddulph et a1. (1959) reported no such steroid increase in peripheral plasma l7-hydroxycortico- steroid concentration in anesthetized dogs during hypoxia (5% oxygen in inspired gas). Analysis of lumbo-adrenal vein plasma has demonstrated that hyperoxia produced by breathing 20 100% oxygen (Marotta et al., 1965) and hypoxia resulting from 10% inspired oxygen (Hirai et al., 1963; Marotta et al., 1963; Marotta et al., 1965) both result in significant increases in the release of l7-hydroxycorticosteroids in dogs. Anichkov et a1. (1960) found that the adrenocortical response to hypoxia disappeared after either hypophysectomy or sinus nerve section. They suggested that the adreno- cortical response to hypoxia is regulated by hypoxic carotid chemoreceptor stimulation which provides input into the hypothalamic-adrenophypOphyseal system for ACTH release. Richards and Stein (1957) demonstrated that hyper- capnia also induces adrenocortical activation for 17- hydroxycorticosteroid release. They found that inspired gas mixtures containing over 10% CO2 results in maximal activation of l7-hydroxycorticosteroid output into the adrenal venous blood of dogs. They concluded that the response was mediated centrally since hypOphysectomized dogs did not exhibit increased adrenal steroid output even after 20% carbon dioxide administration, while the adrenal cortex was still capable of responding to exogenous ACTH. In 1957, Richards infused hydrochloric acid into anesthetized dogs and noted a drOp in arterial pH and PaCOZ’ and a marked elevation of the l7-hydroxycorticosteroid con- Centration in the lumbo-adrenal vein. When sodium bicarbon- ate was infused, arterial pH and P increased, but 17- aco2 hydroxycorticosteroid release also increased. Lau and Marotta (1970) reported that adrenocortical activation in 21 dogs was primarily due to oxygen lack and secondarily due to blood acid-base changes. They also demonstrated that intact carotid and aortic body innervation was necessary for such adrenocortical activation. Hormonal Effects on Respiratory Excitability In 1915, Hasselbalch and Gammeltoft reported that the alveolar partial pressure of carbon dioxide (PACOZ) decreased in women during pregnancy, and promptly returned to normal non-pregnant values at the end of gestation. They also showed that the PAC02 of women was depressed during the luteal phase of the menstrual cycle. Plass and Oberst (1938), and Goodland et a1. (1954) have confirmed these findings and reported an increased alveolar ventilation during pregnancy. Loeschcke and Sommer (1944) studied the respiratory responses of pregnant women to inspired C02. Carbon dioxide response curves (minute ventilation plotted as a function of PACOZ) were shifted to the left during pregnancy, indicating a decreased stimulus threshold for C02. However, the slope of the CO2 response curve during pregnancy was not significantly different from that of the non-pregnant controls, indicating that pregnancy did not alter the respiratory sensitivity to PAOOZ‘ In 1958 Antonio and Lyons used the ventilatory ratio (CO2 induced minute ventilation divided by resting minute ventilation) as an indicator of respiratory excitability, and found that this 22 ratio was higher in pregnant than non-pregnant women, suggesting that pregnancy does increase central respiratory responsiveness to hypercapnic drive. These data implicate progesterone as the hormone which depresses the stimulus threshold for respiratory responsiveness to inhaled CO Using both pre-pubertal and 2. post-menOpausal women, it was demonstrated that pregnanediol injections decreased the PACOZ for six hours after treatment (Heerhaber et al., 1948). Lyons and Antonio (1959) found that progesterone injections given to men and post-menopausal women resulted in both increasing respiratory excitability and in increasing the sensitivity of the central respiratory control center, when evaluated in terms of a carbon dioxide response curve. In 1966, Huang and Lyons reported that the decreased respiratory response threshold to CO2 was not mediated through central chemoreceptors since progesterone treatment induced no significant alterations in CSF pH. The action of progesterone in lowering the arterial CO2 tension (PaCOZ) is clinically useful for treatment of emphysema (Tyler, 1960) and alveolar hypoventilation syndrome of the obese (Lyons and Huang, 1968). Other hormones besides progesterone and pregnanediol have been shown to affect respiration. Koepchen (1953) demonstrated that men treated with cortisone acetate exhib- ited a depression of alveolar PCO2 lasting for approximately 24 hours. He was unable to detect any changes in PACOZ 23 after treatment with testosterone. In 1954, Koepchen et al. reported that ACTH treatment caused a decrease in PAC02 which lasted for about thirty hours after intramuscular injection. In the view of his earlier work, Koepchen concluded that the ACTH effect was secondary to that directly attributable to adrenocortical activation. MATERIALS AND METHODS Animals Three, three year old non-pregnant goats (2 Nubians; 1 Togenberg) whose body weights ranged from 45 to 60 kg were used during the period between July 13, 1970 and March 27, 1971. The animals had been surgically prepared with exter- nalized carotid loops two years prior to testing. The goats were trained to stand in a restraining stanchion and to wear a respiratory mask and valve assembly while in a thermally controlled chamber. Throughout the time of the trials, the animals were housed in sheltered pens and fed a diet of hay and water ad Zibitum. Experimental Conditions Once inside the environmental chamber, the animal was confined in an angle iron stanchion equipped with a vinyl sling which both supported the weight of the goat's trunk and effectively restricted its movement (Figure 1). Chamber temperature was monitored by a tele-thermometer (Yellow Springs Instrument.Co., model 43 TD). Temperature control was maintained using Thermistemp Temperature Con- troller (Yellow Springs Instrument Co., model 71) which 24 25 regulated the pumping of hot or cold water through convec- tive heat exchangers located in front of fans within the chamber. Chamber temperature was adjusted to 25°C 1 0.5°C through the summer until November lst, at which time growth of winter fur necessitated lowering the controlled environ— mental temperature to 20°C in order to remain within the animals' thermoneutral zone.1 A respiratory mask and valve assembly (Lloyd Valve by Warren E. Collins, Inc.) was connected to flexible respiratory hosing to allow collection of expired gas and administration of inspiratory gases consisting of room air plus 21% O in 76.5% N 2 2 2 2 in 74% N2. A 150 liter Douglas bag (Warren E. Collins, Inc.) was employed as an inspiratory buffer and mixtures of 2%% CO , and 5% CO 2 plus 21% O reservoir for the administration of the test gases from high pressure gas cylinders. In view of the findings of Lambert- son et a1. (1961) that pulmonary equilibrium was achieved 7 minutes after beginning CO2 administration to dogs, data collections in this study were made after the goats had breathed each gas mixture for 10 minutes 1Unpublished data characterizing seasonal fluctua- tions in thermoneutral environmental temperature zones of the goat have been previously determined in this lab by W. F. Hofman, G. D. Riegle and B. E. Watkins. Fig. 1. 26 Environmental chamber for data collection. Each goat was positioned in a restraining stanchion (RS) equipped with a vinyl sling (SL) to restrict its movement. Chamber temperature was monitored using a thermister probe (TP) connected to a tem- perature controller (TC) which governed the pumping of water from hot or cold baths (HB/CB) through convective heat exchangers (CH) located in front of circulation fans (F). Inspiratory gas mixtures (IG) were administered through the respiratory mask and valve assembly (RMV). The expired gas was passed through a gasometer for the determination of minute ventilation (VE); a sample of this gas was analyzed for 02 and C02 content. The carotid loop (CL) was catheterized (CA) to monitor heart rate (HR) and blood pressure (BP), and to obtain anaerobic blood samples (BS). Respiratory frequency was obtained using a mercury-in-rubber strain gauge (SG) around the animal's abdomen. A thermocouple inserted into the goat's lower colon was used to monitor rectal temperature (TR). 27 ZOFOUJJOO 5.40 «on. mwmidio AdkzmzzomSZm wanna-u I 8.192112 e u>.~oo.~o 2 0. Eat it 1 2.2 . mo\m.._ 2 28 Experimental Design All experiments were performed at the same time of day to avoid variability which might be due to circadian changes in plasma adrenocortical steroid levels. Ninety CO2 response curves were performed in this study, where two CO2 response curves were determined from one animal on any experimental day. For the generation of each CO2 response curve, the animal was positioned in the environmental cham- ber at 8:30 A.M. Following a 90 minute adaptation period, the respiratory mask and valve assembly was fitted on the snout and the animal was given an additional 30 minutes to adjust to breathing room air through the mask. During the ensuing 15 minutes, four data recordings were made at 5 minute intervals, followed by the drawing of an anaerobic arterial sample (Figure 2). The animal was then given 2%% CO2 as inspired gas for 10 minutes. Four additional data recordings followed by a second arterial blood sample were then obtained. After the goat breathed 5% CO2 for 10 min- utes, the last data recordings were made, followed by the drawing of the third blood sample. Subsequent to a 90 minute recovery period, a second CO response curve was 2 generated as described above. The experimental design included hormone treatment regimes of ACTH and dexamethasone, with therapy durations of both 12 and 36 hours for each hormone (Figure 2). All animals were allowed at least a two week recovery period 29 — A. Generation of CO2 response curve: ! inspire 24% inspire 5% +inspire air IIfI CO2 +< C02-——1 ' ' ‘ ‘ T ' 4 ii A ’I '7’? 1 1% A ' ‘ l i i i 0 min 30 35 40 45 55 60 65 70 80 85 9095 min ‘ ‘ I I I arterial arterial arterial blood blood blood sample sample sample *Record: V E, FEOZI FECOZI HR, BP' f, TR L B. Control: lst CO2 response 2nd CO2 response curve curve 0 hr +14 hr +3 hr +44 hr C. Twelve hour treatments: ACTH and DEXAMETHASONE lst CO2 response 2nd CO2 response inject inject . curve curve L_ I I f T I -12 hr -3 hr 0 hr +14 hr +3 hr +44 hr . Thirty-six hour treatments: ACTH and DEXAMETHASONE inject inject lst CO2 response 2nd CO2 response inject l inject l L + + :36 hr I -13 hr I 0 hr +142l hr +3 hr +44 hr -24 hr -3 hr curve rve _A_ FIGURE 2 TREATMENT SCHEDULES 30 between the completion of one hormone treatment and the initiation of another. The 12 hour ACTH treatment group received intramuscular injections of 80 I.U. ACTH2 at 12 and 3 hours before the respiratory studies. To evaluate possible temporal effects of the treatment, a 36 hour ACTH group receiving similar injections at 36, 24, 12, and 3 hours pre-experiment was included in this study. A second series of experiments utilized intramuscular injections of 24 mg of dexamethasone suspended in corn oil to determine whether the respiratory effects caused by ACTH were due directly to ACTH or to subsequent adrenal activation. Dexamethasone treatment periods were of 12 and 36 hours, with similar injection intervals as described for the ACTH regimes. Data obtained from the animals during each of the hormone treatments were compared to data generated from control animals which did not receive hormone therapy. These uninjected control animals were deemed suitable since they did not exhibit response characteristics distinguish- able from those receiving corn oil injections at 12 and 3 hours before CO2 inhalation. 2The ACTH preparation was Depo-ACTH(§N developed by the Upjohn Co. for respiratory corticotropin injection. 31 Measured Variables Rectal temperature was continuously monitored by means of a copper-constantan thermocouple encased in a polyethylene catheter and inserted approximately 10 cm into the lower colon. The thermocouple was referenced to an ice bath and calibrated with an accuracy of 0.1°C as displayed on a strip chart recorder (Hewlett-Packard, model 7100 BM). Respiratory frequency (f) was recorded using a variable resistance mercury-in-rubber strain gauge affixed to an elastic band which was strapped around the animal's abdomen. Respiratory movements induced changes in strain gauge resistance which led to a matching electrical bridge (model 270 plethysmograph; Parks Electronics Laboratory) and was displayed on a Mosley strip chart recorder (model 7100 BM) . The carotid catheter consisted of a 20 gauge needle connected to a multi-fit stopcock manifold (Becton-Dickenson, model 3170) by means of a length of "Intramedic" polyethylene tubing with an inside diameter of 0.034". The manifold served both as a blood sampling site and as a relay to a pressure transducer (Stathan, model P 23 AC, Hatorey Products). Signals from the transducer were amplified through a D.C. preamplifier (Grass Instrument Co., model 5 P 1 K) for calibrated read-out of mean arterial blood pressure and heart rate on a strip chart recorder (Mosley, model 7100 BM). 32 The expired gas volume was collected for timed intervals at atmospheric pressure and temperature, and saturated with water vapor (ATPS), by passing the gas through a respiratory gasometer (Max Plank Institute of Physiology). Minute ventilation (Vt) was then computed in one of the following manners: P - P (H20) 273 + T ' _ ' B T R VE (BTPS) — VE (ATPS) X P _ P (H O) W (l) B A 2 where: VE (BTPS) = minute ventilation (l/min) at body temperature and pressure, and saturated with water vapor Vt (ATPS) = minute ventilation (1/min) at ambient temperature and pressure, and saturated with water vapor PB = barometric pressure (mm Hg) PT (H20) = partial pressure of water vapor at ambient temperature (mm Hg) PA (H20) = partial pressure of water vapor at body pressure (mm Hg) TR = rectal temperature (°C) T = ambient temperature (°C). . . PB - PT (H20) 273 v STPD = V ATPS —— 2 E I ) E ( ) x 760 273 + T ( ) where: 33 Vt (STDP) = minute ventilation (l/min) at 0°C, 760 mm Hg, and free of water vapor VE (ATPS) = minute ventilation (1/min) at ambient temperature and preSsure, and saturated with water vapor PB = barometric pressure (mm Hg) PT (H20) = partial pressure of H20 vapor at ambient temperature (mm Hg) T = ambient temperature (°C) The fraction of expired carbon dioxide (FECOZ) was measured by passing a continuously flowing sample of the expired gas through a Mead CO analyzer (Harvard Apparatus 2 Co., model 2000) which had been calibrated with gases of known CO2 composition as measured on a Haldane gas analyzer. Similarly, the fraction of expired 02 (F302) was determined by channeling the expired sample through a Beckman paramag- netic oxygen analyzer (model C 2). At the end of each period of breating a gas mixture, an anaerobic arterial sample was drawn from the stopcock manifold into a 5 cc heparinized syringe and promptly sealed with a syringe cap filled with mercury. The blood samples were stored in an ice water bath for subsequent determina- tion of arterial pH, PaCOZ' and bicarbonate (HCO3') concen- tration. Arterial pH measurements were made from a micro- electrode chain attached to a Radiometer pH meter (Radiometer Co., model 27). In determining Paco2 and bicarbonate concen- trations, the samples were allowed to equilibrate with gases of known CO2 tensions in a Radiometer Microtonometer 34 (Radiometer Co., model AMT-1). Values for HCOB‘ concentra- tions in mM/l and for PaCOZ were determined by plotting the anaerobic and equilibrated pH's on a Siggard Anderson (1962) nomogram. Calculated Parameters From measurements of minute ventilation and the fractional composition of expired gas, one can calculate various respiratory quantities. Tidal volume (VT) defines that amount of gas expired in one breath, and is calculated as: v = _E. (3) where: VT'= tidal volume (ml BTPS) VE = minute ventilation (m1 BTPS) f = respiratory frequency (breaths/min) Respiratory dead space (VD) was determined to be 239 i 19 ml (BTPS), which included both the goat's own physiological dead space plus that in the mask and reSpim ratory valve. This quantity was calculated by using the Bohr dead space formula: EEcoz - PaCOZ /(PB‘ PHZO) (4) —v D T FIco2 Pacoz /(PB P320) 35 where: VD = dead space (ml BTPS) VT = tidal volume (ml BTPS) EECOZ = fraction of expired CO2 FICO2 = fraction of inspired CO2 PaCOZ = partial pressure of arterial CO2 (mm Hg) P3 = barometric pressure (mm Hg) PH 0 = partial pressure of H20 vapor at body 2 temperature (mm Hg) Alveolar ventilation (VA) is a term that describes that amount of gas actually flushing through the alveoli in one minute, and is calculated as: V = (V A T - VD) x f (5) where: VA = alveolar ventilation (ml/min BTPS) VT = tidal volume (ml BTPS) VD = ventilatory dead space (m1 BTPS) f = respiratory frequency (breaths/min) Determinations of oxygen consumption (V02) under standard conditions of: 0.0°C, 760 mm Hg and free of water vapor (STPD); were calculated only from the air breathing phase of each experiment because the fraction of inspired oxygen in the CO2 rich gases was not precisely known. The derivation formula is presented as follows: 36 e—VE O - —————-—— F - F F - F 6 E 2 2 2 02 where: V02 = oxygen consumption (ml/min STPD) VE = minute ventilation (ml/min STPD) FIO = fraction of inspired oxygen 2 = fraction of e ired carbon dioxid Ecoz XP e F302 = fraction of expired oxygen Estimations of CSF PC02 were derived from data of Pappenheimer et a1. (1965) which indicated that in goats similar to those of this study, CSF PCQ2 was higher than Pac02 by nine mm Hg for arterial CO tensions between 30 2 and 50 mm Hg. These findings provided the following conversion formula: CSF PCO2 = + 9.0 mm Hg (7) Pa€02 The values of arterial bicarbonate concentrations in mM/l were multiplied by a conversion factor of 1.11 (Cantarow and Schepartz, 1967) in order to express arterial bicarbonate as mM/kg H20. Cerebrospinalfluid bicarbonate concentration was then calculated by employing the relation between arterial and CSF bicarbonate in mM/kg H O, which was formu- 2 lated for unanesthetized goats by Fencl et a1. (1966). The correlation is as follows: CSF HCO3' = 11.3 + 0.352 x arterial HCO3' (8) 37 where: HCO3‘ = bicarbonate concentration in mM/kg H20 Cerebrospinal fluid pH could then be calculated from the Henderson-Hasselbalch equation. The CSF HCO3' con- centration in mM/l, as used in the Henderson-Hasselbalch equation, was found to be not measurably different from that bicarbonate concentration expressed as mM/kg H20 (Cantarow and Schepartz, 1967; Potts and Parry, 1964). Assuming a CSF pK of 6.126 and the soluability coefficient of 0.0314 for the CSF PCO2 (Fencl et al., 1966), the equa- tion appears as: CSF HCO3‘ CSF pH = 6.126 +1og (0.0314)TCSF P002) (9) where: CSF PCO2 = partial pressure of CSF CO2 (mm Hg) CSF HCO ' 3 CSF bicarbonate concentration (mM/l) Statistical Analysis Eighteen CO response curves were obtained for each 2 treatment group. The mean and standard error of the mean for each measured or calculated quantity was determined. Analysis of variance between groups was determined utilizing Student's "t" test, critical to a 5% significance level. All statistical computations were performed on an Olivetti- Underwood Programma 101 computer. RESULTS The mean values obtained for the measured variables of any control animal were not significantly different from those of any other control animal for all inspired gas mixtures. This uniformity suggested using comparisons among test groups rather than among individual animals. Response Changes Due to C02 Inhalation Mean arterial blood pressures, heart rates and rectal temperatures are shown in Table 1. The mean arterial blood pressure of control and both ACTH treated groups was unaffected by breathing C02, while both dexamethasone treat- ment groups showed a higher mean blood pressure when breathing 5% CO2 than when breathing air. Heart rate and rectal temperature of control and all hormone treatment groups were unaffected by various inspired CO2 contents. Table 2 contains mean values of respiratory fre- quency, V and VA for the various hormone treatments at the T different inspiratory gas mixtures. Tidal volume and VA were significantly greater in all groups while inhaling 24% CO2 than when breathing room air, and were also greater at the 5% CO2 level than when the animals breathed either room air or 24% C02. Respiratory frequency was generally not affected by C02 inhalation. 38 39 Tables 3 and 4 contain data concerning arterial and CSF PCOZI pH and bicarbonate concentrations for each hormone treatment while breathing various levels of C02. A11 treat- ment groups exhibited significantly greater arterial and CSF PCO2 values at the 5% CO2 level than during air breath- ing, while the control and the 12 hour ACTH group showed significantly greater PaC02 and CSF PC02 levels while breathing 24% CO2 than when breathing room air. For all treatments, both arterial and CSF pH values while breathing 5% CO2 were significantly lower than when breathing either room air or the 24% CO2 gas mixture. Arterial and CSF bicarbonate concentrations were not affected by C02 inhalation. Response Differences Between Hormone Treatment Groups Blood Pressure, Heart Rate, Rectal Temperature and Oxygen Consumption The data in Table 1 suggest that dexamethasone appears to affect blood pressure in that the mean arterial blood pressures of both dexamethasone therapies were sig- nificantly elevated above those of control and of both ACTH treatments at all inspired CO2 levels. None of the hormone treatments affected the animals' heart rates. There was a significant depression of rectal temperature below control in the 36 hour treatment regimens of both ACTH and dexa- methasone for every inspired gas mixture (Table 1), while 40 oxygen consumption (Table 5) was not significantly affected by any of the hormone treatments. Respiratory Frequency, Tidal Volume and Alveolar VentilatiOn While breathing 5% CO respiratory frequency, V 2' T and VA for all hormone treatment groups were consistently elevated above the corresponding control values, though this difference was not significant in every case (Table 2). Although tidal volume in all treatment groups was elevated above control during the 24% CO inspiration, this differ- 2 ence was only significant for the 12 and 36 hour ACTH treat- ments. Alveolar ventilation in the 12 hour ACTH group was significantly greater than control while breathing 24% C02. As the animals breathed room air, the hormone treatments did not significantly affect respiratory frequency, VT or VA' The hormone injections similarly had no affect on respira- tory frequency while breathing 24% C02. Arterial PCQ2, pH and Bicarbonate Concentration The mean control Paco2 levels were consistently greater than those of all hormone treatments other than the 36 hour ACTH regime during' each level of inspired CO2 (Table 3). Control PaCOZ values were significantly greater than those of the 36 hour dexamethasone treatment group at all inspiratory gas levels, and greater than the 12 hour dexamethasone group's Pac02 values when the animals breathed 24% CO2 and 5% C02. The mean control values for PaC02 were 41 greater than those of the 12 hour ACTH group while breathing room air or 24% C02. The tabular data also indicate that the mean values of the 36 hour ACTH group were in every case greater than control and all other treatment groups while breathing any gas mixture. Though not significantly dif- ferent from control, the 36 hour ACTH treatment resulted in Paco2 levels which were significantly greater than those of both dexamethasone groups at essentially every test gas, and also greater than the 12 hour ACTH Paco2 values when the goats breathed 24% CO The data further demonstrate that 2. the 36 hour dexamethasone treatment's Paco2 levels were significantly lower than in the 12 hour ACTH group during the inspiration of room air and 5% C02. The arterial bicarbonate concentration (Table 3) of the 36 hour ACTH treatment was significantly elevated above control and all other treatment groups for each inspiratory gas mixture. Also, the 12 hour ACTH group exhibited a bicarbonate level significantly greater than control while breathing air. The arterial pH data (Table 3) show that mean control pH values were consistently lower than those of all hormone treatments for every test gas. Control pH values were significantly lower than those of either of the ACTH treatments at each inspiratory mixture, and lower than the 36 hour dexamethasone treatment's arterial pH values while breathing room air and 24% C02. Though not statistically different from control during any inspired gas, the arterial 42 pH of the 12 hour dexamethasone group was lower than that of the 36 hour ACTH treatment while breathing all test gases. The 12 hour dexamethasone group resulted in arte- rial pH values significantly lower than those of either the 12 hour ACTH or 36 hour dexamethasone treatments while breathing air and 24% CO Table 3 also shows that the 2. arterial pH levels of the 12 hour ACTH group were depressed significantly below those of the 36 hour ACTH group when the animals inhaled room air and 5% C02. Cerebrospinal Fluid Pcoz, pH and:Bicarbonate Hormone-induced variations in estimated CSF PCOz (Table 4) were necessarily similar to those measured changes occurring in PaC02° At every inspired CO2 level, the mean control values of CSF PCO2 were greater than the CSF PC02 levels resulting from all hormone treatments other than the 36 hour ACTH group. Control CSF PCQZ was significantly greater than that of the 36 hour dexamethasone treatment at all inspired CO2 levels, and was greater than that of both the 12 hour ACTH and 12 hour dexamethasone therapies while breathing 24% CO The standard errors of the 36 hour ACTH 2. CSF PC02 values were so large that though mean CSF PCO2 levels were greater than those of control or any other treatment group for every inspiration gas, the differences were not generally significant. 43 Because of the procedure for estimating CSF HCO3‘, differences in estimated values of CSF bicarbonate concen- tration among all treatment groups (Table 4) were similar to those variations of arterial HCOB‘ across treatments. The most noticeable difference between therapies is that the 36 hour ACTH group exhibited CSF bicarbonate levels greater than those of all other groups at every level of inspiratory C02. During air breathing, the 12 hour ACTH group's CSF HCO ’ level was also significantly greater than 3 control. The calculated CSF pH of the control group was significantly lower than that of all hormone treated groups for essentially every test gas (Table 4). Aside from this, the only CSF pH difference among the treatments was that the 12 hour dexamethasone pH was significantly lower than that of the 36 hour dexamethasone group while breathing room air and 24% coz. Relationships Between Alveolar ventilation and Blood and CSF Chemistry Figure 3, plotted from the data in Tables 2 and 3, illustrates CO response curves which were obtained both 2 for control animals and for those receiving hormone treat- ments. The curves are generated for each test group by plotting VA on the ordinate as a function of the correspond- ing PaCOZ at various levels of hypercapnic drive to respira- tion. Each curve is drawn through three points representing 44 values obtained during the inspiration of room air-and gas mixtures of 24% CO plus 21% O in 764% N and 5% CO2 plus 2 2 2’ 21% O in 74% N . The 36 hour dexamethasone group's 2 2 responses were significantly shifted to the left of control at all three inspiration gases, while the 12 hour dexa— methasone treatment resulted in a significant leftward shift of the response curve during both levelsof induced hyper- capnic drive to respiration. The curve corresponding to the 12 hour ACTH treatment was also significantly shifted to the left of the control response at the room air and 24% CO2 points. The 36 hour ACTH treatment resulted in a response curve which tended to lie to the right of control, but this shift was not significant at any point. Figure 4, depicting data from Tables 2 and 3, graphically represents the relationships between mean VA and arterial pH values obtained for all test groups while breath- ing the three gas mixtures. The response curves of both ACTH groups and of the 36 hour dexamethasone treatment were significantly shifted to the left from the curve generated by the control group at each level of inspiratory C02. On the other hand, the response curve described by the 12 hour dexamethasone treatment did not result in any significant deviation from that of control for any of the test gas inspirations. In Figure 5, alveolar ventilation is plotted as a function of CSF H+ concentration (data from Tables 2 and 4). A11 hormone treatments tended to exhibit response curves 45 which were shifted to the left from that of control. The 36 hour dexamethasone treatment resulted in a response which was significantly to the left of control for all three gas mixtures. Values for both the 12 hour dexamethasone and 12 hour ACTH treatments are shifted to the left from corre- sponding controls at the room air and 24%'CO2 inspiration points. 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HGOHHMHHusm> HMHom>Hm wow 00 muoumnHmmcH 0cm mpcmsumwnu waoEHon Mo muommmm .« mHnme H1mEdHo> HmoHu 48 .Hm0.0vmv mHDHxHE mmm pmHHmmcH mchcommmHHoo um .xma H: «H Eonw ucmanMHU >HucmoHMHcmHm .Am0.0vmv musuxHE mmm pmuHmmcH mCHUGOQmeHoo um mBUH H: mm Eoum ucmHmMMHp hHucmoHMHcmHma .Hm0.0vmv musuxHE mmm UmHHmmcH mcHUcommeHoo um mBUH H: «H Eoum ucmHmMMHc hHucmoHMHcmHmU .Hm0.0vmv mHDHxHE mam pmHHmmcH mchcommmHHoo um Houucoo Eoum ucmHmMMHp >HusmoHMHcmHmm .Am0.0vmv Hcmfiummuu wcoEHon mEmm mcHHsp UOHHmQ «00 mn« Eoum ucmHmMMHU mHHGMOHMHcmHmn .Hm0.0vmv unmaummnu meoEHos mEMm msHHsc GOHHwQ HHm Soon Eoum uanmMMHc mHucmoHMHamHmM .zmm H mmsHm> smmzm .zmm H mmsHm> some uo«m mx\ZE~ .Smm H mmsHm> :mmE «mm 852 n m 0H0.0 H 55m.5 a H m H00.0 H 0mv.5 a H m00.0 H mvv.5 0H .xmo H: 0m 0 n m HH0.0 H 00m.5 o m H00.0 H 00v.5 U m m00.0 H H«v.5 0H .xmo H: «H m H n m «H0.0 H 0Hv.5 H 0H0.0 H 5mv.5 m H «H0.0 H m5¢.5 0H mBUH H: mm H n w H00.0 H m5m.5 H 0H0.0 H Hmv.5 H 000.0 H mvv.5 0H mBUH H: «H Q m H00.0 H mmm.5 000.0 H vam.5 000.0 H «H¢.5 0H Honusou mm HHHHmHHH U m.H H «.m« U m.H H 0.0« o 0.H H 0.0« 0H .xma us on o 0.H H H.m« U m.H H 0.0« U 0.0 H v.m« 0H .xma an «H H «.H H «.5m m H 5.0 H H.5m m H H.H H m.0m 0H mBUH H: mm 0.0 H m.0m 5.0 H «.0m H «.0 H m.0m 0H mBUH H: «H 5.0 H 0.0« 5.0 H 5.0« m.0 H H.m« 0H Honusoo .Hmon HMHHGHHH O m H n m 5.0 H 0.HH U H 0.H H 0.0m O m H m.H H H.0m mH .xwa H: 0m 0 H a m m.0 H H.0v U H H.H H 5.HH «.H H 0.Hv 0H .xmo H: «H m «.m H m.mm m 0.m H H.0H m.« H 0.mv 0H mBUH H: mm b m 5.0 H 0.5v H m m.0 H «.«v H m.0 H m.HH mH mBUH an «H n m 0.0 H 0.mv m H.H H m.mv 5.0 H m.«v 0H Houucoo «cum HMHHmHHH Noo Hm Noo me HHH soom mHmHHe msHmmm Hams I mo .02 nummua mGOEHom mmw pmHHmmsH a ucmfimunmmmz HmonoHOHmmsm «OU « Hum HMHumuHm 0cm «.mumsonHMOHn HmHumuHm H. m HHHHmHHm so 00 mnoumuHmmsH paw musmfiummuu mcoEHon mo muowmmm .m mHnma 49 .A00.0vmv mHDHxHE mmm wmnHmmCH mchcommmHHoo Hm .H00.0vmv mHSHxHE mmm UmHHmmcH mchaommmHHoo Hm .A00.0vmv mHsuxHE mmm UmHHmmaH mCHvsommmHHoo Hm .H00.0vmv mHsuxHE mam pmHHmmsH msHpsommmHHoo .x00 HQ NH mBUH M: 0m mBUH HQ NH Hm Houusoo .H00.0vmv HamEHmmHu waofiuoz mEmm msHusc UOHHmm «00 0«« .H00.0vm0 usmfiummuu waofinon 08mm mcHHsp UOHH mm HHM Boon Eonm EOHm Eoum Eoum Eoum Eoum HcmHmHHHo HamHmHHHe HcmHmHHHe HcmHmHHHo HcmHmHHHc HcmHmHHHo HHHcmoHH HcmHm HHHHHOHHHamHm HHHnmoHHHcmHm HHHcmoHHHcmHm a U m H Q m HHucmoHHHamHm HHHHHOHHHamHm .zmm H mmsHm> smmzm .Smm H mmSHm> came Ho«m 0x\zEH .zmm H mmsHm> same “mm 881 H Q m 000.0 H mm«.5 O H m 000.0 H «5«.5 O H 000.0 H 50«.5 0H .xmo H: 00 H Q m 000.0 H 0««.5 H 000.0 H 0m«.5 H 000.0 H m0«.5 0H .xmo H: «H m 0H0.0 H mH«.5 0H0.0 H 0m«.5 H HH0.0 H 05«.5 0H mBUH Hg 00 H Q m H00.0 H 0H«.5 H 000.0 H 0m«.5 H 000.0 H 00«.5 0H mBUH HQ «H Q m 000.0 H «H«.5 6 000.0 H 00«.5 000.0 H mm«.5 0H HOHHCOU mm th U 0.0 H 0.H« U 0.0 H 0.H« U 0.0 H 0.H« 0H .Nmn HS 00 U 0.0 H 0.H« U 0.0 H 0.H« U 0.0 H 0.H« 0H .xmo H: «H m H 0.0 H 0.0« m H «.0 H 0.0« m H 0.0 H H.0N 0H mBUH HQ 00 0.0 H 0.«« «.0 H 0.H« H H.0 H 0.«« 0H mBUH Hz «H «.0 H 5.H« «.0 H 0.H« H.0 H 0.H« 0H HOHHGOU mHMGOQumon 000 o m H Q m 0.0 H 0.00 U H 0.H H 0.00 m H 0.H H H.50 0H xmn H: 00 Q m 0.H H 0.00 H 0.H H 5.00 «.H H 0.00 0H .xmo Hz «H m m «.m H m.«0 m.« H 0.50 m.« H 0.00 0H mBDH HQ 00 Q m 0.0 H 0.00 H m H.0 H 0.Hm 0.0 H 0.00 0H mBOH Hz «H m 5.0 H 0.00 m 0.0 H 0.00 0.0 H m.Hm 0H HOHHQOU moom 000 «00 00 «00 0«« HHH Eoom mHMHHB mEHmmm Hume mo .02 lummna mGQEHom mmo umHHmmaH a Hamsmusmmoz HMOHmoHOHmmsm mmU H.«Oum mmu @wumHsono so «00 wuoumuHmmcH Usm mus mum mmo can «.mHMGOQHmoHQ mfiummuu mcofiuon mo muommmm .0 mHQMB 50 Table 5. Effects of hormone treatments on oxygen consumption1 Number 9 Treatment of Trials 02 ml/min (STPD) Control 18 241 1 18 12 hr ACTH 18 256 i 25 36 hr ACTH 18 247 i 39 12 hr Dex. 18 236 i 20 36 hr Dex. 18 320 i 41 1Mean values 1 SEM. Fig. 3. 51 Effect of hormone treatments on the ventilatory response to induced changes of arterial PC02' These data represent C02 response curves for all test groups, which relate alveolar ventilation (VA) on the ordinate with arterial carbon dioxide ten- sion (PaC02) on the abscissa. Each curve is drawn through three points corresponding to VA and Paco values obtained while breathing room air and 2 modified air mixtures containing 24% C02 and 5% C02. The control response is represented by circles connected with a solid line. Crosses linked by dashed lines correspond to values obtained during the ACTH treatments, with the 12 and 36 hour treatment durations labeled in the figure. The reSponses obtained during the 12 and 36 hour dexa- methasone treatments are likewise identified by circles connected by dashed lines. 52 Noon. H.554 to 80256 9.682. 8 $2880 5023.5? ME 20 mpzmsEmE 020.200: “.0 Bake m 0039.0 ~88 Hmlmm 00 0.0 ow 0.H mzomdrkmidxmo 1:0 IP04 7...: .m: 85.28 "leg 0 mm \ 8%.. O. '“!“‘/'1 VA 0. ON 0N Fig. 4. 53 Effect of hormone treatments on the ventilatory response to induced changes in arterial pH. These data represent response curves for all test groups, which relate alveolar ventilation (VA) on the ordinate with arterial pH on the abscissa. Each curve is drawn through three points corre- sponding to VA and arterial pH values obtained while breathing room air and modified air mixtures containing 24% C02 and 5% C02. The control response is represented by circles connected with a solid line. Crosses linked by dashed lines cor- respond to values obtained during the ACTH treat- ments, with the 12 and 36 hour treatment durations labeled in the figure. The responses obtained during the 12 and 36 hour dexamethasone treatments are likewise identified by circles connected by dashed lines. 54 I0 44.1953 2. mUOZHIo ow0302_ O... mmzoawmm >¢OF MI... 20 mhzm—zhHmmh MZOZmOI mo Bun—mm 0 0000: :a 3222 one. 02. one. oi. «0.H. 1:. mi. 03H mm 0 VA. .o. 1 I m. .m. u x x \ x ”120memeng Io \ x x 1.54. "I...“ o o. .6528 "lo. 0N Fig. 5. 55 Effect of hormone treatments on the ventilatory response to estimated changes of CSF H+ concen- tration. These data represent response curves for all test groups, which relate alveolar ventilation (VA) on the ordinate with a combined abscissa of CSF pH and CSF H+ concentration. Each curve is drawn through three points corresponding to VA and CSF pH values obtained while breathing room air and modified air mixtures containing 24% C02 and 5% C02. The control response is represented by circles connected with solid lines. Crosses linked by dashed lines corre- spond to values obtained during the ACTH treatments, with the 12 and 36 hour treatment durations labeled in the figure. The responses obtained during the 12 and 36 hour dexamethasone treatments are like- wise identified by circles connected by dashed lines. 56 T: “.8 mo $025.6 822:3 o» mmzoammm £183.ng MI... 20 mHZMEPHMKP mZOEmOI no howumm m mmaoi on: 9:2,. Ta .13 mm 8 on no. 0... c.» an an» arm “.8“: 3H m: in VA. 40. all m. u in. x \ ION \\ X \ 2.8050on00 Tb u. H \ EST... O\ s\ _OL.—:OOHIIO o. .3 DISCUSSION Heart rate was unaffected by either the hormone treatments or by hypercapnia induced with inspired CO2 (Table 1). The mean arterial blood pressure of the dexa- methasone treatments was significantly greater than that of both control and the ACTH injected animals for all inspira- tory gas mixtures (Table l). The maintenance of blood pressure associated with ACTH injections agrees with the findings of Sambhi et a1. (l963),who reported increased cardiac output, decreased peripheral resistance and un- changed mean arterial blood pressure following ACTH treat- ment to normal and hypotensive subjects. The increase in blood pressure during dexamethasone therapy, with no sig- nificant alteration in heart rate, suggests that the dexa- methasone effect was brought about by increasing peripheral resistance or by increasing cardiac output. It is possible that dexamethasone increases peripheral resistance by exhibiting the known glucocorticoid action to potentiate the vasoconstrictive response of adrenergic drugs (Zweifach, 1960). Although reasons for the different effects of the two types of hormone treatment on blood pressure are not apparent, they may be related to specific biological actions 57 58 of natural glucocorticoids and dexamethasone or to differ- ences in effective hormone dosages available to responsive tissue. Oxygen consumption (Table 5) was not significantly affected by any of the hormone treatments. Control rectal temperature was consistently greater than that exhibited by either of the 36 hour treatment groups for all levels of inspired CO (Table 1). These results indicate that the 2 hormone treatments did not significantly alter whole body metabolic heat production as evidenced by the unchanging V02, and may have induced the development of slight hypo- thermia as indexed by a depression of rectal temperature. Since both ACTH and dexamethasone treatments resulted in lowering rectal temperature without changing V02, the effect may have been caused by glucocorticoids to activate certain thermoregulatory heat loss mechanisms without measurably altering heat production as indexed by unaltered VOZ. Since VA was generally not significantly elevated above control in either of the 36 hour treatment groups at any level of inspired CO (Table 2), the increased heat loss was likely 2 mediated cutaneously rather than by means of increasing evaporative water loss. The data indicate that the hypo— thermic response begins at a time between 12 and 36 hours after hormone treatment initiation. The inhalation of gas mixtures rich in CO2 resulted in increased PCO2 and decreased pH in blood and cerebro- spinal fluid. It is generally accepted that, in the absence 59 of hypoxia, the increased respiratory activity induced by C02 breathing results to a much greater extent from stim- ulation of central respiratory chemoreceptors than from those at the peripheral level (Gemmill and Reeves, 1933; Lambertson, 1968). Alterations in the level of respiratory activation were measured in terms of changes in VA, pre- sumably reflecting fluctuations of the efferent output from the medullary respiratory control center. The respiratory control center, which is assumed to consist of the inspira— tory and expiratory neuron groups in the medulla generates electrical signals which travel along such avenues as the phrenic nerve (Lourenco et al., 1966) to regulate activity of respiratory musculature. Differences of VA between con- trol and hormone treated animals at specific arterial or CSF levels of pH and PC02 were assumed to reflect hormonal effects upon the activity of the respiratory control center. Tidal volume and alveolar ventilation both increased with elevated inspiratory CO2 content (Table 2). On the other hand, respiratory frequency was unchanged with in- creased levels of inspired C02, indicating that the increase in VA was primarily due to elevated VT. These findings are in agreement with data reported by Lambertson et a1. (1961) and Mitchell et a1. (1963). At the 5% level of inspired C02, all hormone treatment grdups had greater mean values for f, VT and VA than those of the control group. Although most of the increases in f, V T and VA were not significantly greater than the corresponding control values, the consistency of 60 the elevations provides support that both hormones may be involved in the alteration of these respiratory responses. Although breathing CO had no effect on the arterial 2 HCO3' levels in any of the groups tested, comparisons of bicarbonate levels between treatment groups showed that only the 36 hour ACTH treatment group had arterial bicarbonate concentrations different from the control group (Table 3). The bicarbonate concentration of the 36 hour ACTH treatment averaged 7.0 mM/kg H 0 higher than that of any other groups, 2 indicating development of metabolic alkalosis in these animals. However, the extent of this development was vari- able as evidenced by the high standard error associated with these bicarbonate measurements. The 36 hour ACTH treatment regime likely induced maximal adrenocortical activation to lead to the development of a Cushingoid syndrome which in- cludes the development of metabolic alkalosis. This treat- ment presumably resulted in the release of appreciable amounts of adrenocortical steroids, all of which possess some mineralocorticoid activity. It has been shown that mineralocorticoids induce an increased renal loss of potas- sium. Migration of hydrogen ions into intracellular sites may then have occurred to compensate for the potassium lost. The result of these electrolyte shifts would be the develop- ment of extracellular metabolic alkalosis (Nocenti, 1968). This decrease in extracellular hydrogen ion concentration would then tend to drive the bicarbonate buffer equilibrium toward the formation of increased bicarbonate. 61 + -> ‘I' "' C02 + H20 + H2CO3 + H + HCO3 (9) The mean PaCOZ at each test gas mixture was greater in the control group than in any of the hormone treatments except the 36 hour ACTH group (Table 3). This relationship implies that the respiratory response to arterial PCO2 was less in the control than in all hormone treatment groups other than the 36 hour ACTH treatment, in that the control animals exhibited a greater tolerance to arterial PCO2 for each inspiratory gas mixture than did the hormonally treated groups. Similar decreases in alveolar PC02 have been reported during pregnancy and after pregnanediol treatment (Hasselbalch and Gammeltoft, 1915; Heerhaber et al., 1948; Goodland et al., 1954). The mean Paco2 values of the 36 hour ACTH group were higher than those of any other treat- ment group. It is possible that the elevated PaCO2 levels at all gas mixtures in the 36 hour ACTH group were related to the development of metabolic alkalosis during this treat- ment, where the increased blood bicarbonate concentrations would cause a shift of the bicarbonate buffer equilibrium to increase the arterial tension of C02. The tolerance of elevated PaC02 by this group suggests that the arterial CO2 tension does not in itself constitute a sufficiently strong respiratory stimulus to decrease the Pacoz level. Figure 3 illustrates the relationships between VA and arterial PCO2 values which were obtained for control and hormone treated groups while breathing the three test gases. 62 The leftward shift of both dexamethasone treatment groups and the 12 hour ACTH group from that of control suggests that those hormone treatments acted to increase respiratory excitability. Increased respiratory excitability can be visualized as a parallel leftward displacement in a respi- ratory response curve (Figures 3, 4, and 5), and defined as an increased VA above control when associated with a spe- cific level of arterial or CSF pH or PC02° These indica- tions of hormone-induced increased respiratory excitability concur with similar CO2 response curve alterations that have been reported following injections of cortisone (Koepchen, 1953), ACTH (Koepchen et al., 1954) or progesterone (Huang and Lyons, 1966), and during pregnancy (Loescheke and Sommer, 1944; Lyons and Antonio, 1959). On the other hand, the mean 36 hour ACTH response fell to the right of the control group response at each inspiratory gas, suggesting a decreased respiratory excitability in this group. However, the vari- ability of the 36 hour ACTH responses was so great that the shift toward respiratory depression was not significant. The difference in ventilatory excitability between the 12 and 36 hour ACTH regimens was unexpected. The observation that respiratory excitability was not different from control in the 36 hour treatment group may be due to the existence of metabolic alkalosis in this group. By shifting the bicarbonate buffer equilibrium toward elevating PaCOZ' this metabolic condition could act to mask any hormone-induced 63 alterations in respiratory excitability when VA is gauged as a function of PaC02° Arterial pH in all groups decreased with increasing inspired CO2 (Table 3; Figure 4). The decrease in pH is directly associated with the increase in PaCO2 which shifts the bicarbonate buffer equilibrium to favor the formation of hydrogen ions in the blood. The average values of arte- rial blood pH for the control group were significantly lower than those of any treatment group except the 12 hour dexa- methasone therapy. The findings indicate that the respira- tory excitability of these hormone treatments is greater than control for every tested level of hypercapnic drive to respiration when arterial pH is used as an indicator of respiratory drive. The response curve described by the 36 hour ACTH treatment tended to be shifted further to the left of control than were the curves of the other hormone treat- ments. The presence of metabolic alkalosis in the 36 hour ACTH group presumably causes a displacement of hydrogen ions intracellularly from‘vascular stores, thereby increasing arterial pH values above those that would be recorded if the treatment group were not in an acid-base imbalance. As has been noted, the arterial values of pH and Paco2 became appreciably altered during the development of metabolic alkalosis (Table 3, Figures 3 and 4). An attempt was thus made to estimate values for CSF pH, PC02 and HCO3' levels from the corresponding arterial data in order to evaluate the effects of CSF hydrogen ion concentration on 64 the central chemoreceptor sites. To make possible the calculation of CSF pH, estimations of CSF PC02 and CSF HCO3' concentrations were derived from the data of Pappen- heimer et a1. (1965) and Fencl et a1. (1966), respectively, who simultaneously compared these aspects of CSF and arte- rial composition in goats similar to those used in this study. Upon increasing inspired CO2 content, the calculated CSF Pco2 was elevated as shown in Table 4, supporting the hypothesis that with increased inspired CO2 concentrations, substantial diffusion of CO2 occurs from the blood across the "blood-brain" barrier and into the cerebrospinal fluid. It was observed that the control group maintained a higher CSF PCO2 than did all treatments other then the 36 hour ACTH group, suggesting a difference between control and hormone treated groups in the central respiratory response to CSF PCOZ' It is again assumed that the difference between the 36 hour ACTH group's CSF PC02 and that of the other treat- ment groups is related to the metabolic acid-base imbalance occurring during the 36 hour ACTH treatment. The calculated CSF pH values during CO2 inhalation indicates that the CSF pH while breathing 5% CO was signif- 2 icantly lower than corresponding pH values during room air and 24% CO2 breathing for all treatments. While breathing air and 24% C02, the mean CSF pH of the control group was lower than the mean calculated CSF pH of all treatment groups. Although the measured mean arterial pH of the 65 36 hour ACTH treatment was higher than other groups at all inspiratory gas mixtures, the computed hydrogen ion concen- trations in CSF during the 36 hour ACTH treatment were not significantly different from the other hormone treated groups. These data demonstrate a deviation of hydrogen ion concentration alterations between the CSF and vascular com— partments associated with metabolic alkalosis. Although metabolic alkalosis acts to increase blood pH, it does not measurably alter CSF pH from that occurring in the other hormonally treated groups. The increase in respiratory excitability as indexed by CSF pH (Figure 5) was consistent across most treatment groups, with only the room air and 24% CO2 points of the 36 hour dexamethasone treatment different from those of the 12 hour dexamethasone treatment group. Generally, respira- tory excitability increased by a similar magnitude above control for all hormone injection groups while breathing any gas mixture. The increased respiratory excitability can be quantified with the observation that all hormone treatments decreased the effective set-point of the output from the respiratory control center by a calculated hydrogen ion concentration of about 3.0 nM/kg H 0. These results are 2 inconsistent with those of Huang and Lyons (1966) who reported that no alterations in CSF pH occurred in response to progesterone treatment of dosages sufficient to increase VA' control group was 2.6 liters/min per nM H+ while the slopes The slope of the ventilatory response curve for the 66 of all hormone treated groups averaged 2.7 liters/min per nM H+, which was not significantly different from the con- trol slope. This relationship suggests that the sensitivity (change in VA for given change in CSF H+ concentration) of the respiratory control center in response to changes of CSF pH was not measurably altered by increased gluco- corticoids. The data suggest that arterial blood PCO2 or pH are not totally reliable indicators of the effective degree of respiratory activation when the test animals experience a metabolic acid—base disturbance. The development of meta- bolic alkalosis from mineralocorticoid activity in the 36 hour ACTH group presumably altered the bicarbonate buffer equilibrium toward increasing CO formation. This increased 2 CO2 would result in biasing the PaCO2 ventilatory response curve to the right of values that would have been expressed under normal acid-base conditions (Figure 3). The intra- cellular migration of hydrogen ions to replace the loss of potassium ions during metabolic alkalosis would then lower the arterial pH below normal levels. This decrease in arterial pH may then be associated with a shift in the arterial pH related ventilatory response curve (Figure 4) further to the left than would have occurred under normal conditions (Nocenti, 1968). These results indicate that arterial pH or PaCOZ cannot themselves account for the observed alterations in respiratory activity during the development of an abnormal metabolic acid-base status. 67 Fencl et a1. (1966) demonstrated that the respira- tory response to inhaled CO in unanesthetized goats was a 2 single function of cisternal CSF hydrogen ion concentration, regardless of metabolic acid-base status. In the present study, all the hormone treatments were associated with a shift of the respiratory.excitability, producing an increase in ventilation above control group values for all levels of CSF pH. On the other hand, analysis of the respiratory response to arterial pH or Paco2 changes did not produce a similar relationship capable of explaining ventilatory alterations on the basis of changes in these arterial quantities. The data suggest that cisternal CSF hydrogen ion concentration estimations appear to be the preferred index of respiratory excitability in that the relationship between VA and estimated CSF pH was alone able to reflect response changes of consistent direction and magnitude for all glucocorticoid elevating treatments. The alteration in respiratory excitability, as indexed by CSF pH, was similar after both 12 and 36 hours of ACTH stimulation, illustrating response consistency even during the development of metabolic alkalosis. The similarity of the responses of the 12 and 36 hour ACTH and dexamethasone groups to CSF pH indicates that the effectiveness of ACTH was secondary to its recognized action of activating the adrenal cortex. The comparability of the 12 and 36 hour data from both treatments implies that 68 the alteration in respiratory excitability occurred within the first twelve hours after initiation of hormone treatment. The glucocorticoid effect on respiration may be brought about by directly increasing the activity of the medullary respiratory control center to increase respiratory excitability by approximately 3.0 nM H+/kg H20. The gluco- corticoids may instead function on the central or peripheral chemoreceptors to increase their level of afferent neural input to the respiratory control center, thereby altering respiratory excitability. It is also possible that gluco- corticoids alter the strength of an input to the respiratory control center from some source other than that of the chemoreceptors. It may be that glucocorticoids do not in themselves directly increase respiratory excitability. These adreno- cortical hormones may in fact function by increasing the availability of certain nervous tissue chemical mediators or transmitter substances. As feedback inhibitors of corticotrOpin releasing factors, glucocorticoids are recog- nized to effect the synthesis and/or release of central neuro-transmitters (Vernikos-Danellis, 1965). It is con- ceivable that the glucocorticoid effect on respiratory excitability could involve similar mechanisms. Gluco- corticoid effect on adenosine triphosphate available for neuro-transmitter substance synthesis, or hormone effects on the secretion or uptake of such neuro-transmitter 69 substances as norepinepherine (Maas and Mednieks, 1971) could ultimately be responsible for increasing the level of respiratory activation. SUMMARY This investigation was designed to assess the influence of ACTH and glucocorticoids on reSpiratory activ- ity. AdrenocorticotrOpin and dexamethasone treatments were separately administered in 12 and 36 hour therapy durations to resting, unanesthetized adult female goats. Hormonal effects on the alteration of respiratory responses and blood chemistry were measured during various levels of hypercapnic respiratory drive, while concomitant changes in cisternal CSF chemistry were calculated from the arterial data. Oxygen consumption was unaffected by hormone treat- ments, while the rectal temperatures of the 36 hour ACTH and 36 hour dexamethasone therapies were both lower than control. The results suggest that glucocorticoid treatment of more than 12 hours resulted in the develOpment of a hypothermic effect, presumably by activating a thermoregulatory heat loss mechanism because metabolic heat production as indexed by Vbz was unchanged. Another observed effect of the hormone therapies used in these studies was that the 12 and 36 hour dexa- methasone treatments resulted in an elevation in arterial blood pressure with no alteration in heart rate. This increased blood pressure presumably reflects an increase 70 71 in either peripheral resistance or cardiac output during dexamethasone administration. Analysis of the arterial data suggested that the 36 hour ACTH treatment induced the development of metabolic alkalosis which was presumably due to mineralocorticoid activity of the natural hormones of the adrenal cortex. The electrolyte shifts associated with this metabolic state caused significant increases in arterial bicarbonate con- centration, CSF bicarbonate concentration, PaC02 and arte- rial pH above corresponding values in the other treatment groups. On the other hand, metabolic alkalosis did not significantly alter the calculated CSF pH from values observed in the other hormone treatment groups. T’ f and VA for all hormone treated animals inspiring 5% CO2 were higher than the corresponding Mean values of V control means. The arterial PC02 data indicated that all treatments except the 36 hour ACTH group (which experienced metabolic alkalosis) exhibited greater respiratory excit- ability than control since the vA during these hormone therapies was greater than that of control at all Pac02 levels measured. ventilatory excitability gauged as a function of arterial pH was likewise inconsistent in that all treatment groups other than the 12 hour dexamethasone regime displayed significant increases in respiratory excit- ability above control at all levels of arterial pH. When VA was evaluated in terms of calculated CSF hydrogen ion concen- tration, it was found that all treatment groups exhibited an 72 increased respiratory excitability consistently greater than control by 3.0 nM/kg H20. 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