OIMB QP 321 .H33 1987 CHANGES IN MYOGLOBIN AND LACTATE DEHYDROGENASE IN MUSCLE TISSUES OF A DIVING BIRD, THE PIGEON GUILLEMOT (CEPPHUS COLUMBA), DURING MATURATION by LISA MARIE HAGGBLOK A THESIS Presented to the Department of Biology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Haster of Science June 1987 APPROVED: Dr. Robert C. Terwilliger ;' ii j :1j! I j- 1 i .1 ." I iii An Abstract of the Thesis of Lisa Marie Haggblom for the degree of in the Department of Biology to be taken Master of Science June 1987 I I +1 ! i Title: CHANGES IN MYOGLOBIN AND LACTATE DEHYDROGENASE IN MUSCLE TISSUES OF A DIVING BIRD, THE PIGEON GUILLEMOT (CEPPHUS COLUMBA), DURING MATURATION Approved: Dr. Robert C. Terwilliger Physiological adaptations of heart and pectoralis muscle tissues to diving-induced hypoxia were compared among three stages of maturation of the Pigeon Guillemot (Cepphus columba); chick, fledgling, and adult. Myoglobin ~oncentration increne~~ fr~~ fledb:~ng to adult i.eart ~na pectoralis muscles, while myoglobin polypeptide expression changed between chick and fledgling pectoralis muscles. Total lactate dehydrogenase (LOH) activities in pectoralis muscle increased from chick to fledgling. The ratio of LDH-5 to LDH-l, 2, 3, and 4 1n f~edgling pectoralis was greater than that in chick and adult pectoralis. Hematocrits and blood oxygen capacities increased from chick to fledgling to adult. Pigeon Guillemots, as they mature from chicks to adults, become better adapted to exerClse and diving. Aerobic metabolism is preferred in adult heart and pectoralis muscles, although pectoralis muscles potentially resort to anaerobic metabolism during temporary periods of hypoxia. / VITA NAME OF AUTHOR: Lisa Marie Haggblom PLACE OF BIRTH: Brooklyn. New York DATE OF BIRTH: December 26. 1962 GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon Metropolitan State College. Denver DEGREES AWARDED: Master of Science. 1987. University of Oregon Bachelor of Science. 1984. Metropolitan State College AREAS OF SPECIAL INTEREST: Avian and Mammalian Diving Physiology PROFESSIONAL EXPERIENCE: Laboratory Assistant. Department of Biochemistry/Biophysics. University of Colorado Health Sciences Center. Denver. 1983-85 iv J t Research Assistant. Department of Cardiovascular-Pulmonary Research. University of Colorado Health Sciences Center. Denver. 1984-85 AWARDS AND HONORS: Golden Key Honor Society Membership. 1984-1987 Colorado Scholars Award. 1983-84 ACKNOWLEDGEMENTS I thank Professors Nora and Robert Terwilliger for their enthusiasm and encouragement throughout all phases of this study; Dr. Janet Hodder and Michael Graybill for their invaluable assistance in the field; Daniel Varoujean for his advice on dissection procedures; Dr. Robert Becker for the key to comprehending International Enzyme Units; and Dr. Robert C. Cohen for his editorial assistance. I also thank the OIKB students. staff. and Charleston residents who supported me in various ways throughout the duration of this research. I specially thank Wendy Lou Manley. who. by sharing her love and appreciation of life. enhanced my own. This research was supported by NSF grant DKB-8511150 to N. B. Terwilliger and R. C. Terwilliger. v t I r i t Chapter 1. II. III. IV. APPENDIX A. B. TABLE OF CONTENTS INTRODUCTION••••••••••••••••••••••.••••..••••••••••••••. MATERIALS AND METHODS ••••••••••••••••••••••••••••••••••• Anima 1 Co llect ion •••••••••.•••••••.••.•••••••••••••••. Blood Parameters •••••••••••••••••••••••••••••••••••••• Myog lobin ..•.••••••.•..•.••••...................•..•.. Preparation of Myoglobin Extract •••••••••••••••••••• Myoglobin ABsay••••••••••••••••••.•••••••••••••••••• Myoglobin Electrophoresis ••••••••••••••••••••••••••• Lactate Dehydrogenase (LDH) ••••••••••••••••••••••••••• Preparation of LDH Solution ••••••••••••••••••••••••• Enzyme Assay •••••••••••••••••••••••••••••••••••••••• Electrophoresis •••••••••••••••.••••••••••••••••••••• Statistical Analyses ••••••••••.••••.••.••••••••••••••• RESULTS ••••••••••••••••••••••••••••••••••••••••••••••••• HeDl()g lobin .•..•.•.••.•..•............................. Kyoglobino, ;. .:.ijej •• -G ••••••••••••••••••••• Concentration ••••••••••••••••••••••.•••••••.•••••••• Electrophoresis ••••••••••••••••••.••••••.••••••••••• Lactate Dehydrogenase .........•....................... Act ivity .•..•.•••.....•............................. Electrophoresis•.........•..................•....... Enzyme Kinetics •••••••••••••••••.••••••.••.••••••.•• DISCUSSION••.•••.•••••••••••••.•..•••.•••..•...••.••..•• HeUl()g lob in ••••••••••••••••••••.•••••.••••••••••.•••.•• Myog lobin ••••••••••••••••••••.••••••••..•••••••••••.•• Lactate Dehydrogenase ••••••••••••••••••••••••••••••••• SUlDDl8.ry ••••••••••••••••••••••••••••••••••••••••••••••• GROWTH CURVE OF PIGEON GUILLEMOT CHICKS (1982, 1983) •••• HEMATOCRITS, HEMOGLOBIN CONCNS, AND OXYGEN CAPACITIES OF DIVING AND NON-DIVING ANIMALS •••••••••••••••••••••••• vi Page 1 8 8 9 10 10 11 11 12 12 12 13 13 14 14 14 / 14 15 17 17 17 20 23 23 24 26 30 32 33 vii C. MYOGLOBIN CONCNS (G/IOO G WET WEIGHT TISSUE) IN MUSCLE TISSUESa OF DIVING AND NON-DIVING ANIMA.LSb • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 34 D. TOTAL LDH ACTIVITIES (ru/G WET WEIGHT/KL) OF MUSCLE TISSUESa IN DIVING AND NON-DIVING ANIKALSb.............. 35 E. Km ESTIMATES (PYRUVATE, MIL) OF CHICKEN, OX (BEEF), AND RABBIT HEART AND SKELETAL MUSCLES (ADULTS).............. 36 BI BLIOGRA.PHY • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 37 ; I I / 'I viii LIST OF TABLES Table Page 1. Age, Sex, and Weight of the Six Pigeon Guillemots Collected for This Study................................. 9 2. Hematocrits, Hemoglobin Concns, and Oxygen Capacities of Chick, Fledgling, and Adult Pigeon Guillemots(±Stand. Dev.).......................................... 14 3. Myoglobin Concn. (g/100 g Wet Weight) in Heart and Pectoralis Muscles of Pigeon Guillemot Chick, Fledgling, and Adult (u·S).......................................... 15 4. Km Estimates (Pyruvate, MIL) of Heart and Pectoralis (Pect.) Muscles of Pigeon Guillemot Chick, Fledgling, and Adult. . • • •• • • . • • • • . • . . . . • . • • . • • • • • . • • • • •• • . • • • • . . . • . • • • . • 22 / / ,.,. ix LIST OF FIGURES Figure Page 1. Heart Myoglobin in Pigeon Guillemot Chick (C), Fledgling (F), and Adult (A), and Sperm Whale Myoglobin (SWM). {Loading Volumes - 3, 5 microliters (ul).)...................................... 16 2. Pectoralis Myoglobin in Pigeon Guillemot Chick, Fledgling, and Adult, and Sperm Whale Myoglobin. {Loading Volume R 10 u1.)............................... 16 3. Total LDH Activity in Heart and Pectoralis Muscles of Pigeon Guillemot Chick, Fledgling, and Adult (n-5). (pH 7. 4J 25°C.)....................................... 18 4. Heart (H) and Pectoralis (p) LDH Isozymes in Pigeon Guillemot Chick, Fledgling, and Adult. {Loading Volumes c 2, 5, 10 ul.)................................. 19 5. Pectoralis LDH Isozymes in Pigeon Guillemot Chick, Fledgling, and Adult. {Loading Volume R 10 u1.)........ 19 6. Velocity versus SuhULLat~ (pyr&vate) Concentratio; of LDH in Pigeon Guillemot Chick, Fledgling, and Adult (n-2). (pH 7.4, 25°C.) ••...•••••••.••.•.•••••••••••••••••••••.. 21 .i CHAPTER I INTRODUCTION The physiology of exercise and diving has been a topic of great interest to biologists (Scholander, 1940; Andersen, 1966; Ridgway and Scronce, 1969; Kooyman, 1972; Kerem et al., 1973; Hochachka et al., 1975; Jones, 1976; Butler and Jones, 1982; Mill and Baldwin, 1983; Deshpande et al., 1984; Castel1ini et al., 1985; Grigg et al., 1986; Hochachka, 1986). One main focus of these studies is ho~ Bnin~Js respond to lowered oxygen tension (hypoxia). Studies on a wide range of animals have shown numerous behavioral, physiological, and biochemical adaptations to hypoxia (Irving et al., 1941; Andersen, 1966; Vesell, . 1906; Cohen, 1969; Ridgway and SCl.unce, 1969; Kooyman'," 1972, 19~5; Kerem et al., 1973; Hochachka and Storey, 1975; Butler and Jones, 1982; Castellini et al., 1985). Diving induces hypoxia; adaptations to hypoxia include apnea, bradycardia, and peripheral vasoconstrictions--collectively known as the diving response (Scholander, 1940; Catlett and Johnston, 1974; Zapol et al., 1979; Murphy et al., 1980; Butler and Jones, 1982; Butler and Woakes, 1984; Hochachka, 1986). These adaptations insure that oxygen 16 reserved primarily for the organs most critical to sustaining life--the brain, heart, and lungs (Bron et al., 1966; Angell-James and Daly, 1969; Butler and Jones, 1971; Andersen and Blix, 1974; Jones et al., 1979). 1 / At the molecular level, skeletal muscles are specially adapted to tolerate hypoxic conditions (Scholander et al., 1942; Simon et al., 1974; Hochachka and Storey, 1975; Castellini and Somero, 1981; Castellini et al., 1981; Hochachka, 1985). Myoglobin facilitates the diffusion of oxygen into cells (Wittenberg, 1959; Hemingsen, 1963; Kreuzer, 1970; Wittenberg et al., 1975; Cole, 1983), but may also act as an oxygen storage molecule in muscle tissue. A relatively high myoglobin concentration in muscle tissue is one adaptation to hypoxia. Diving animals have higher concentrations of myoglobin in their muscle tissue than non-diving animals. This oxygen store is available during periods of hypoxia (Theorell, 1934; Kendrew et al., 1954; Ridgway and Johnston, 1966; Lenfant, 1969; Blessing, 1972; Weber et aI, 1974; Castellini and Somero, 1981; Mill and Baldwin, 1983). Another mechanism of adaptation is the enhancement of glycolytic pathwRys. Musclf' contr'lction8 are fueled by adenolline t:riphosphate (ATP), which is produced from lipids, carbohydrates, and amino acids. When oxygen supply to the muscles is constant, during periods of steady exercise, lipids are oxidized in the Krebs cycle, and ATP is produced. When oxygen supply is limited, during periods of rapid, short-term ~ercise, ATP is synthesized from carbohydrates, such as glycogen, V1a glycolysis. This way, lipid reserves can be used for periods of normoxia, and glycogen stores can be saved for temporary periods of hypoxia. Anaerobic glycolysis requires nicotinamide adenine dinucleotide (NAD+) to produce ATP; NAn+ is available via the 2 following reaction: Pyruvate + NADH LDH +Lactate + NAD 3 Lactate dehydrogenase (LDH, E. C. 1.1.1.27) is the enzyme involved in this reversible reaction (Cahn et al., 1962; Kaplan and Cabn, 1962; Dawson et al., 1964). There are five LDH isozymes, and each isozyme is a tetramer consisting of two types of subunits, Hand M. Heart muscle has mostly LDH-l, thus LDH-l is comprised of H subunits. Skeletal muscle has mostly LDH-5, thus LDH-5 is comprised of M subunits. LDH-2, 3, and 4 consist of intermediate ratios of the subunits. Most tissues have a combination of the five isozymes (Kaplan et aI, 1960; Cabn et al., 1962; Muller and Baldwin, 1978). LDH-5 mainly converts pyruvate to lactate in the skeletal muscle, and is not inhibited by high pyruvate concentrations. Lactate is then .. transported by the blood to the heart muscle, where LDH-l, which is inhibited by bigh concentrations of pyruvate, converts lactate to pyruvate. Pyruvate is then oxidized in the Krebs cycle and ATP is produced (Dawson et al., 1964; Kaplan, 1964; Andersen, 1966; Lehninger, 1982). As a result of these differences in isozyme properties, beart ~scle can maintain aerobic metabolism, while skeletal muscle can resort to anaerobic metabolism during hypoxia (Cabo et al., 1962; Dawson et al., 1964; Kaplan, 1964; Kaplan et al., 1968; Markert and Masui, 1969; Hochachka and Storey, 1975; Baldwin et al., 1978; Castellini and Somero, 1981) • / , I Not only 16 the type of LDH Isozyme present In the tissue indicative of hypoxia tolerance, but the total activity of LDH In the tissue can indicate its metabolic tolerances. If the total LDH activity is relatively high, due to a high activity of LDH-5, the tissue can, most likely, metabolize anaerobically (Fine et al., 1963; Pesce et al., 1964; Castellini et al., 1981). Muscle tissues of comparative functions of diving and non-diving animals show dramatic differences in myoglobin concentrations and LDH Isozyme patterns and activities. Pinnipeds (Blix et al., 1970; Storey and Hochachka, 1974; Behrisch and Elsner, 1980), cetaceans (Blessing, 1972; Caste1lini et al., 1981), and aquatic reptiles and amphibians (Gatten, 1985; Grigg et al., 1986) have been compared with rodents (Epstein et a1., 1964; Caste11ini et a1., 1981), primates (Vesell, 1965; Hochachka and Storey, 1975), ungulates (Pesce et al., 1964; Blix et al., 197 5), and terrestrial cernivcn:,: (Tf~(;ell and Pool, 1966. Cao~.:lliIii et al., 1981). Within Class Aves, some skeletal muscles of diving birds--such as penguins--have higher myoglobin concentrations (Weber et al., 1974; Mill and Baldwin, 1983) and higher ratios of LDH-M to LDH-H isozymes (Wilson et a1., 1963; Markert and Masui, 1969; Deshpande et aJ., 1984) than skeletal muscles of non-divers--such as chickens (Cahn, 1964; Pesce et al., 1964). A comparison of the same muscle tissues of diving and non-diving individuals of the same species is also instructive (Markert and Masui, 1969; Weber et al., 1974). This comparison includes possible genetic and behavioral, as well as physiological, adaptations to hypoxia. The same tissues can be compared from the non-diving to diving stages of 4 /' maturation (Weber et al., 1974). Also, different tissues can be compared at the same stages of diving Dlaturation (Markert and Hasui, 1969; Weber et al., 1974). Birds of the family Alcidae, which includes Murres, Hurrelets, Puffins, Auklets, Razorbills, Dovekies, and Guillemots, are especially adapted to the marine environment. Alcids breed along coastlines during late spring and summer, and they winter at sea; they are primarily pelagic. They dive for their food, using their wings for propulsion, but little is known of their pelagic diving habits. Thoreson and Booth (1958), Drent (1965), Cody (1972), Sealy (1975), Asbirk (1979), Cairns (1981), and Rasmussen (1983), have studied aspects of the breeding and behavioral biology of Alcids. Follet and Ainley (1976) have described prey items of Pigeon Guillemots (Cepphu~ columba) during their nesting season, and Piatt and Nettleship (1985) have accumulated diving depths of several species of Ale ids from gill'-nets l.i.tBhor~ and offsllC're. These depths range from 50 meters (m) for Black Guillemots (Cephhus grylle) to 200 m for Common Murres (Uria aalge). Piatt and Nettleship (1985) have also recorded dive durations of 112 seconds (s) for Black Guillemots diving in water 35-45 m deep, and Pigeon Guillemots have been observed t9 dive for 20 sec In water less than 10·m deep (personal observation). As of now, few studies have focused on the possible physiological or biochemical adaptations of Alcids to hypoxia induced by diving. (Bradley and Threlfall, 1974; Kostelecka-Hyrcha, 1987). The diving species in this study is the Pigeon Guillemot (Cepphus columba), which breeds along the west coast of North America. Pigeon Guillemots reach sexual maturity at approximately 3 years of age, and 5 ,-/ are sexually monomorphic. They feed on benthic fish and invertebrates. and eat a wide variety of prey items (Thoreson and Booth. ]958; Drent. 1965; Follet and Ainley, 1976). Nest sites are typically located in crevasses and crannies on slopes and cliffs on the coastline (Thoreson and Booth. 1958; Drent. 1965). Man-made structures. such as three-sided cubicles formed by the supporting beams of bridges (Hodder and Graybill. unpublished observation) and abandoned piers (Hodder and Graybill, 1985), also provide nest sites. Both male and female parents feed the chicks at the nest sites. The nestling Pigeon Guillemots are active; however. they do not swim or dive. The adults abandon the nest site a few days before the young fledge, approximately thirty to forty days after hatching. and move offshore. The fledglings leave the nest site and begin diving and feeding as they also move offshore (Thoreson and Booth. 1958; Drent. 1965). In this the~is. I have asked t~e following questions in r~gArd6,to the muscle adaptations of the chick, fledgling, and adult Pigeon Guillemot. First. does the total concentration of myoglobin 1ncrease in the heart and pectoral muscles with maturation of the chick? Second, IS there a change in the composition of myoglobin polypeptide chains expressed at the three stages of maturity? Third. does the total activity of LDH increase in the heart and pectoral muscles as the chick matures to adult? Finally. do the ratios of isozymes in these two muscles change between stages of maturation? I also measured several blood parameters to compare Pigeon Guillemot chick. fledgling. and adult blood with that of other diving and non-diving animals. / 6 / 1 I I ij The findings of this study shall contribute to an understanding of the total dive response of Pigeon Guillemots, as well as other diving birds. 7 / . ; 1 I I CHAPTER II MATERIALS AND METHODS Animal CQII~ctiQn Six Pigeon Guillemots were collected for this study: two adults and two chicks were collected from the Charleston Bridge in Charleston, Oregon, on June 1 and June 29, 1986, respectively; and two fledglings were collected from Sitka Dock in Empire, Oregon, on August 3, 1986 (Table 1, p. 9). Pigeon Guillemots use the supporting beams under these structures as nest sites, which are accessible by boat during high tide. The birds were netted, then killed by quick raps to the skull with a 8 club within approximately 2 minutes (min) of netting. The chest cavity -/ ';as opened' ill~llediately after death, and biood was collected into a heparinized syringe from the heart and aorta. The blood samples and entire bodies were then placed on ice. Upon return to the lab, the blood samples were immediately prepared; the birds were completely dissected; and the heart and pectoralis muscles were removed and stored The ages of the chicks and fledglings were estimated from a growth curve. The curve was obtained from averaging nestling weight versus nestling age data from two years, 1982 and 1983, from nest sites at Sitka Dock (Hodder and Graybill, unpublished observations). (Appendix A.) TABLE 1. Age, Sex, and Weight of the Six Pigeon Guillemot8 Collected for This Study ------------------------------------------------------------------ Collection Maturation Sex Weight Age Date Stage (g) (days) ----------------------------------------------------------------- 6/01/86 adult (1) male 439 unknown 6/01/86 adult (2) male 497 unknown 6/29/86 chick (1) feDis Ie 105 5 6/29/86 chick (2) male 196 9 8/03/86 fledgling (1) unknown 481 32 8/03/86 fledgling (2) fems Ie 506 36 Blood Parameters Hematocrits (Hct) were determined in microcapillary tubes (Sherwood) spun in an IEC microcapillary centrifuge for 3 m1n. The spectrophotometrically after conversion to cyan-met hemoglobin (Drabkin and Austin, 1932; Kampen and Zijlstra, 1961. 1965). Ten microliters of whole blood were added to 3 milliliters (ml) of Drabkins reagent and mixed, then potassium cyanide (KCN) and potassium ferricyanide (1S 3Fe(CN)6) were added to convert the blood solutiol) to the cyan-met form. The absorption (A) of cyan-met hemoglobin was read at 540 nanometers (nm) on a Zeiss PM QII spectrophotometer, and hemoglobin was determined from the relationship: Hb concentration in grams (g) per 100 ml ~ AS40 x 36.4. The oxygen-carrying capacity (02 cap.) of whole blood was calculated from the relationship: oxygen capacity (g/100 ml) ~ Hb x 1.368 (Dijkhuizen et al., 1977). 9 Myoglobin Preparation of Myoglobin Extract 10 I I Approximately 0.1-0.5 g of frozen muscle tissue was homogenized in 3.0 m] ice cold. 0.1 molar (M) sodium phosphate buffer. pH 7.4. for 3 m1D 1n a Waring blender. The homogenate was centrifuged at 12.000 g for 30 min 1D a refrigerated centrifuge (RC2-B Sorval). The supernatant was decanted and saved; the pellet was rehomogenized and centrifuged a second time as described above. The supernatants were then combined to form the crude extract. Muscle tissue can contain hemoglobin as well as myoglobin. In order to measure only myoglobin concentration in the crude extract, the hemoglobin was precipitated out of the extract according to the procedure suggested by Weber et al. (1974). Solid ammonium sulfate was aoded to the ~tl.1df' ~~~t::-acL. whilt: s~.i.::.-iU6. t::> 65% of satul:'&l:ivu. l.ll order to precipitate the hemoglobin. The $upernatant (containing myoglobin) was decanted, and ammonium sulfate was then removed from the myoglobin solution by dialysis against a 0.1M solution of ammonium bicarbonate, pH 8.0. To test the effectiveness of this procedure. the precipitate (containing hemoglobin) was resuspended in the buffer described above. Then both the hemoglobin precipitate and myoglobin supernatant were assayed by column chromatography (Sephadex G-75 gel. 2x92 cm) and spectrophotometry (Zeiss PM QII spectrophotometer). The resulting absorption peaks (measured at 540 nm) from the two fractions ensured that mostly myoglobin remained in the supernatant fraction, and that little myoglobin was precipitated out by the ammonium sulfate. The dialyzed sample was then concentrated (Centricon-10 microconcentrator) and used for myoglobin assays and electrophoresis. (Repetitions of assays were performed on each extract preparation when possible.) Myoglobin Assay The concentration of myoglobin was measured spectrophotometrically as cyanmet-myoglobin. KCN and K3Fe(CN)6 were added to the sample to convert the myoglobin to cyanmet-myog10bin. The absorbance of the cyanmet-myog1obin was read at 540 nm (Reynafarje, 1963) with a Perkin-Elmer Double Beam spectrophotometer and recorder. A millimolar extinction coefficient of 11.0 and a molecular weight of 17,000 for myoglobin (Van Assende1ft, 1970) was used to calculate the myoglobin concentrations, wh;.rp;<: f>---"~-:~~:J ,;/100 g we;' ':IJ:::ig~,:':::f t:~SSUE'. Myoglobin Electrophoresis Polyacrylamide gel electrophoresis (PAGE), and PAGE in the presence of sodium dodecyl sulphate (SDS-PAGE), separate myoglobin from other ~roteins, as well as the myoglobin polypeptide chains from each other. Two methods--7.5%-PAGE with pH 8.9 buffer (Davis, 1964) and 14% SDS-PAGE with pH 8.3 buffer (Laemmli, 1970)--were compared to determine ",hich gave the most complete separation of the myoglobin polypeptide chains. The gels were run at 35 milliamps (rnA) for 8pproxiD~tely 2.5 hours at room temperature; stained overnight with CooID8ssie blue R250 in isopropanol-acetic acid, the stain described by 11 Fairbanks et al. (1971); then destained with 10% acetic acid (Fairbanks et al., 1971). The 7.5%-PAGE did not resolve the polypeptide chains as well as the 14% SDS-PAGE; thus, 14% SDS-PAGE was used for subsequent separations and for qualitative purposes only. Lactate Dehydrogenase (LDH) Preparation of LDH Solution 12 The procedure for preparation of the crude LDH extract was the same as the procedure for the preparation of crude myoglobin extract Enzyme Assay LDH activity 1n solution was determined spectrophotometrically from the rate of oxidation of NADG, 88 pyruvate was reduced to lactate (Pesce et al., 1964). The reaction medium in the cuvette (total volume of 3.0 ml) contained the following: 0.3M pyruvate; 2.13 millimolar (roM) NADH; O.lM sodium phosphate buffer, pH 7.4; and diluted enzyme sample. The change in absorbance of the reaction medium at 340 nm was measured with a Perkin-Elmer double-beam spectrophotometer, using a roM extinction coefficient of 6.22 for NADG (Windholz, 1983). The absorption reading was valid even in the presence of other substances in the cuvette, since the other substances had an absorption of zero at 340 nm. Enzyme activity was expressed in International enzyme units (IU/g wet weight of tissue/ml) (Dixon and Webb, 1964). Pyruvate -2 -5 concentrations ranging from 1.00 x 10 M to 1.00 x 10 M were used to determine the maximum velocity (Vmax) of the enzyme reaction in the tissues (Bergmeyer, et al., 1963). The Michaelis constants (Km values) of the enzyme ~n the various tissues were determined by Lineweaver-Burk plots (Dixon and Webb, 1964). Km values represent the concentration of substrate (pyruvate) at which the enzyme reaches half of its maximum velocity. They provide some measurement of tbe rate at which pyruvate is converted to lactate. Electrophoresis LOB isozymes were resolved with 5.5%-PAGE (Dietz and Lubrano, 1967). The gels were run at 35 mA for approximately 2.5 bours at room otemperature or 4 C, then stained from 45-60 min at room temperature, 13 with the stain described by Markert and Kasui (1969). Tbe isozymes were separated with equal resolution at both temperatures. The extract preparations were subjected to electrophoresis in this manner r.ep~atedly. and tbp. re'8tiv~ darkness of the b8nds'~a3 ~0illpared by visual inspection only. Statistical Analyses Where applicable, 95% confidence intervals, according to the S-test, were calculated for statistical analyses (Snedecor and Cochran, 1980). 14 CHAPTER III RESULTS Hemoglobin The fledglings and adults had hematocrits, hemoglobin concentrations, and oxygen carrying capacities approximately 2-3x higher than those of the chicks (p < 0.050). There were no significant differences between the fledgling and adult blood parameters (Table 2). TABLE 2. Hematocrits, Hemoglobin Concns, and Oxygen Capacities of Chick, Fledgling, and Adult Pigeon Guillemots (± Stand. Dev.) ------------------------------------------------------------------------ Stage of Hct Hb 02 cap. Maturation (%) n (vol %) n (vol %) n ------------------------------------------------------------------------ Chick ]').34 ± 4.6~ c; 6.62 ± 2098 8 9.06 ± ~ I\Q .:l.,,- Fledgling 46.64 ± 6.91 8 16.00 ± 2.20 8 21.89 ± 3.01 8 Adult 50.70 ± 0.74 4 17.70 ± 3.75 4 24.21 ± 5.13 4 Myoglobin Concentration Table 3 shows the myoglobin concentration of the heart and pectoralis muscle tissues for the three age groups--chick, fledgling. and adult. The chick heart had more myoglobin than the chick pectoralis, and the fledgling heart had more myoglobin than the fledgling pectoralis (p < 0.050). There was no significant difference, 15 however, in the myoglobin concentration of the heart and pectoralis muscles of the adult. The concentration of myoglobin was greater in the adult heart and pectoralis than in the two younger stages (p < 0.050). TABLE 3. Myoglobin Concns (g/100 g Wet Weight) in Heart and Pectoralis Muscles of Pigeon Guillemot Chick, Fledgling, and Adult (n e 5) Stage of Maturation Heart Pectoralis ------------------------------------------------------------------------ Chick .32 ± .04 .14 ± .01 Fledgling .20 ± .03 .14 ± .04 Adult .71 ± .11 .65 ± .14 The chick and fledgling pectoralis muscles appeared light red; the adult pectoralis was dark red. The heart muscles of all three stages were deep red. Electrophoresis No difference could be distinguished in the electrophoretic mobility of adult, fledgling, and chick heart myoglobin (Figure 1). The mobilities of the myoglobin of the adult and fledgling pectoralis were similar to one another, and to heart myoglobin, but differed from that of the chick pectoralis (Figure 2). ---1 I AFCAFC 16 I SWM- t;:~.~.,-I sd. .............'.-.~_ ..- __ e-- "..- ,'- • FIGURE 1. Heart Myoglobin in Pigeon Guillemot Chick (C), Fledgling (F), and Adult (A), and Sperm Whale Myoglobin (SWM). (Loading Volumes = 3, 5 microliters, (ul).) A F C t FIGURE 2. Pectoralis Myoglobin in Pigeon Guillemot Chick. Fledgling, and Adult, and Sperm Whale Myoglobin. (Loading volume = 10 u1.) sw M- --- - ~ - .. •~ 'x;... \ - --,. ,,"!''t =:rcrf.' ..'. . '. ~ .,., ~. >. - , .;.< ""," 'j ... - .- • .. • .- .. II I I Activity Figure 3. p. 18. ShOW6 the LDH activities. In IU/g wet weight of tissue/mI. of the heart and pectoralis muscles for the different age groups (pH 7.4. 250 C). Both adult and fledgling had significantly higher LDH concentrations In the pectoralis muscle than the respective heart muscle (p < 0.050). In contrast. the LDH concentration in the chick pectoralis was not significantly higher than in the chick heart. The concentration of LDH in the heart tissue of all stages was similar. However. the LDH concentration was about twice as great in the fledgling and adult pectoralis tissue than in the chick pectoralis tissue. Electrophoresis Figure 4. p. 19 illustrates the electrophoretic p8ttet~ of th~ five isozymes of LDH. LDH-l (H4 ) is the top band seen on the gel. and LDH-5 (M4), is the bottom band, the band which migrates farthe6t towards the anode. The three bands in between, from H4 to M4 , are the following: H4 (LDH-l); H3M} (LDH-2); "2M2 (LDH-3)j HIM3 (LDH-4); and M4 (LDH-5). All five isozymes were present in both heart and pectoralis tissues at all stages, but the heart muscle had predominantly "4 isozymes, and the pectoralis muscle had predominantly M4 isozymes. There was an interesting pattern in the pectoralis isozymes. however, that was not present in the heart isozymes. The heart isozymes appeared to be similar for the chick. fledgling. and adult. but the pectoralis isozymes differed amongst the three age groups (Figure 5). The chick appeared to 17 ,I i \ t 18 25 ~ ".or. ~ OP.,to roll ' 0- "::> 20 -' >- .... > t- 15 \ 0 4 \ :r: \ /" 0 I ...J \, tOO \ ...J 4 t- O t- chlctt STAGE OF MATURATION FIGURE 3. Total LDH Activity in Heart and Pectoralis Muscles of Pigeon Guillemot Chick, Fledgling. and Adult (nc 5). (pH=7.4, 25°C.) / \ I I , LOH-I H P I I I I I I A F C A F C 1=1 I rTI I I I I I ::1 N I£)~ LDH - 5 19 FIGURE 4. Heart (H) and Pectoralis (P) LDH Isozyrnes in Pigeon Guillemot Chick, Fledgling, and-Adult. (Loading volumes = 2, 5, 10 ul.) I AFC~FC LOH - 5 FIGURE 5. Pectoralis LDH Isozymes in Pigeon Guillemot Chick, Fledgling, and Adult. (Loading Volume = 10 ul.) 20 have a similar concentration of isozymes LDH-I through LDH-4. with a higher concentration of LDH-S. The fledgling also had a strong LDH-S band. but there appeared to be more LDH-4 than LDH-3, 2. and 1. The adult pectoralis had predominantly LDH-S, but the difference in concentration between isozyme Sand isozymes 1-4 was not as great as for the chick and fledgling isozymes. Enzyme Kinetics The chick, fledgling, and adult heart enzymes all showed substrate inhibition (pH 7.4, 2SoC). (Figure 6. p. 21) The pyruvate to lactate reaction rate reached a maximum velocity as pyruvate concentration increased, then the reaction rate decreased. The degree of substrate inhibition appeared to be greater in the adult heart than the fledgling and chick heart. In all stages the pectoralis LDH maintained a more conl'ltan~ react5.':\n "atf> ,.fter maximum ~elocity-hadbeE::ll oocained. a-oo tiid not show the substrate inhibition that the heart muscle LDH showed. The Km estimates for LDH, shown in Table 4. were higher for the pectoralis tissue than for the heart tissue for all stages. The Km values appeared to be larger in the fledgling and adult than in the chick for both muscle tissues. 21 ch ick 6·0~.O4.02-01.001 - •.. ::- Ic e fledQling j! .:50 I... I• .40a. ,j "- I .30 I0 II V Ito . 0 0 >" - >- f- ad u It (,) .5 0 ...J .4 UJ > 3 .2 PYRUVATE CONCENTRATION (M J( 10 -3) FIGURE 6. Velocity versus Substrate (Pyruvate) Concentration of LDH in Pigeon Guillemot Chick. Fledgling, and Adult (n=2). (pH=7.4, 25°C). o -Pectoralis. A -Heart. 1 I, ! ~ 22 TABLE 4. 1m Estimates (Pyruvate, MIL) of Heart and Pectoralis (Pect.) Muscles of Pigeon Guillemot Chick, Fledgling, and Adult. Animal Tissue Range Mean n ------------------------------------------------------------------------ Chick heart 5.80 x 10-4 - 1.08 x 10-2 6.51 x 10-3 6 pect. 5.40 x 10-3 - 3.82 x 10-2 1.63 x 10-2 4 Fledgling heart 5.75 x 10-4 - 5.55 x 10-2 2.80 x 10-2 2 pect. 9.31 x 10-2 - 1.38 x 10-1 1.16 x 10-1 2 , Adult heart 1.07· x 10-:l 1.20 .x 10-1 6.54 x 10-2 . 2 pect. 2.86 x 10-1 - 1.05 x 10 0 6.68 x 10-1 2 ------------------------------------------------------------------------ CHAPTER IV DISCUSSION HemoglQbin The hematocrits and 02 capacities of fledgling and adult Pigeon Guillemot blood are relatively high, and comparable to those of other adult Alcids (Bradley and Threlfall, 1974; Kostelecka-Myrcha, 1987). The high blood 02 capacity of the fledglings and adults may be related to their increased activities; on the other hand, the lower hematocrit and blood 02 capacity of Pigeon Guillemot chicks may be related to their relative inactivity at the nest site. The values for Pigeon Guillemot chicks are one half to one third those recorded for Common Murre (Uria aalge) and Dovekie (Plautys aIle) chick& (Bradley and Threlfall, 1974; Kostelecka-Myrcha, 1987). Murre chicks, however, differ from Pigeon Guillemot chicks by swimming with the parent before fledging--an activity which may well be related to high blood 02 capacity. Blood of other diving birds, such as penguins, some ducks, and loons, has a high 02 capacity similar to blood of fledgling and adult Pigeon Guillemots and other Alcids (Bond and Gilbert, 1958; Lenfant et al., 1969; Milsom et al., 1973; Guard and Murrish, 1975; Mill and Baldwin, 1983). Birds that maintain steady speeds during flying or fly for long periods of time, such as pigeons, swifts and hummingbirds, also have relatively high hematocrits and blood 02 capacities (Bond and Gilbert, 1958; Palomeque et al., 1980; Johansen. 1987). Thus./diving 23 1l and sustained flight in birds appears to require similar blood carrying capacities. Shallow-diving pinnipeds and cetaceans have blood 02 capacities similar to Pigeon Guillemot adults--values which are 2-3x those of humans (Harrison and Kooyman, 1968; Horvath et al., 1968; Lenfant et al., 1968; Clausen and Ersland, 1969; Lenfant et al., 1969, 1970; Guard and Murrish, 1975; Palomeque et al., 1980; Hedrick and Duffield, 1986). (Appendix B.) Myoglobin Myoglobin concentration 1n both heart and pectoralis muscle increases during nlsturation of the Pigeon Guillemot from the chick to the adult. The fledglings collected for this study were approximately 32 and 36 days of age, and the adults' age could only be approximated to at least three years old, the age of sexual maturity. The myoglobin concevtret;on may g~adually ip~~ease over maturation from fledgling to sexually mature adult, or it may sharply increase immediately after fledging, when the bird begins to dive for food. If muscle tissue tolerance to hypoxia is correlated with muscle oxygen storage, then muscles with relatively high myoglobin concentrations can tolerate long periods of hypoxia, theroretically (Weber et al., 1974). The heart muscle in the Pigeon Guillemot adult, however, does not tolerate hypoxia, but has as much myoglobin as the adult pectoralis muscle. Perhaps Pigeon Guillemot myoglobin stores oxygen in the pectoralis muscle tissue, allowing the muscle to tolerate hypoxia, while in the heart muscle tissue myoglobin facilitates a continuous oxygen supply. 24 Muscle fibers can be classified into three types. according to George and Berger (1966): red. white. and intermediate fibers. Red (aerobic) fibers have large numbers of mitochondria. high oxidative enzyme activity. and high myoglobin content. White (anaerobic) fibers have fewer mitochondria J higher glycolytic enzyme activity. and low myoglobin content. Intermediate fibers have characteristics of both fibers. Although the distribution of fiber types in the muscles was not determined for the Pigeon Guillemot. the low concentration of myoglobin in the pectoralis of the chick and the fledgling suggests there are predominantly white fibers. During the progression from very light red fibers to dark red fibers in the pectoralis as the chick matures. the myoglobin polypeptides expressed first change. then increase in concentration. It is not unreasonable to suggest that this is correlated with a change from predominantly anaerobic to aerobic metaboli&lli. dud from ?redominantly white t~ red muscle fibers. The adult pectoralis muscle tissue potentially metabolizes aerobically most of the time. and relies on reserve oxygen stores only during infrequent periods of hypoxia. The heart. in all stages of n~turation. however. metabolizes aerobically. based on overall muscle fiber color and u~changing myoglobin polypeptide expression through maturation. Muscle myoglobin concentrations in both tissues of the adult Pigeon Guillemot are intermediate between those of adult pigeon and penguin skeletal muscle tissue. with adult penguin concentrations being the highest of the three (Lawrie. 1950; Weber et al •• 1974; Mill and Baldwin. 1983). Pigeons may use myoglobin mainly for a continuous oxygen supply to highly aerobic tissues. but not for oxygen storage for 2S ....~. 26 potentially anaerobic tissues. Conversely, penguins may use myoglobin mainly for oxygen storage for their anaerobic tissues during diving (Weber et aI, 1974). Thus. the myoglobin concentration in tissues of the adult Pigeon Guillemot are higher than those of a non-diver, and lower than those of an optimal diver like the penguin. Deep-diving seals and whales and highly active rodents, such as rabbits and some bats, have the highest recorded myoglobin concentrations in skeletal muscle tissue, which is approximately 8x that of adult Pigeon Guillemots (Eichelberger, 1939; Scholander, 1940; Perkoff and Tyler, 1958; Blessing and Harschen-Niemeyer, 1969; George et al., 1971; Blessing, 1972; Ohtsu, et al., 1978; Castellini and Somero, 1981). (Appendix C.) Lactate Dehydrogenase The total LDH concentration (213 IU/g ± 43) in skeletal (pectoral) muscle of tht:: a-dult Pigeon Gui l1em.::<: is similar to that :in other diving birds such as the Dabchick (Deshpande et al., 1984). It is only one tenth that reported for penguins (Castellini and Somero, 1981; Mill and Baldwin, 1983) and seals (Castellini and Somero, 1981), both of which are more accomplished divers than the Pigeon Guillemot (Appendix D). On She other hand, penguin skeletal muscle LDH is approximately 3-4x that of cardiac muscle (Castellini and Somero, 1981; Mill and Baldwin, 1983); similarly, Pigeon Guillemot skeletal muscle LDH is approximately 2-3x that of cardiac muscle. This suggests that, although the total enzyme concentrations differ, skeletal and heart muscles have similar metabolic requirements in these two birds. During maturation. the greatest 1ncrease 1n total LDH activity 1n the pectoralis muscle occurs between the chick and fledgling stage (Figure 3). the same period of time when the myoglobin protein expression changes. This increase in LDH is the result of an enhancement mostly of LDH-5. since the fledgling pectoralis has the highest ratio of LDB-5 to other isozymes. LDB-5 is less sensitive to pyruvate inhibition than LDB-l. and therefore can reduce more pyruvate +for HAD regeneration during periods of anaerobiosis (Dawson et al •• 1964; Kaplan, 1964; Everse and Kaplan. 1973; Bochachka, 1980; Mill and Baldwin, 1983). The fledgling pectoralis then should be better able to tolerate hypoxia than the chick pectoralis. As the fledgling matures into the adult, the ratio of LDB-5 to the other isozymes decreases in the pectoralis, but LDB-5 still remalDS the dominant isozyme. The adult pectoralis, therefore, is capable of both aerobic and anaerobic means of iIleti1bo~icm. Heart "!IUscie from 1)1! :;t.::l6.::a of waturat':'onhaii iii.Jstly . LDH-l, which primarily functions to oxidize lactate and recycie pyruvate into the Kreb's cycle for ATP production. Therefore, the heart predominantly metabolizes aerobically (Dawson et al., 1964; Kaplan, 1964; Everse and Kaplan, 1973; Bochachka, 1980; Mill and Baldwin, 1983). The likely conclusion would be that the heart muscle has to function aerobically whether it is in the chick, fledgling, or adult. During the development of Adelie penguin embryos, Markert and Masui (1969) reported a shift in isozyme expression from LDB-I to LDH-4 and 5 in the pectoralis muscle (over a span of approximately 23 days). The heart muscle had consistent LDH-l activity throughout development, but the other isozymes fluctuated in expression until LDH-l, 2, and 3 27 28 persisted at the end of the developmental period. They noted that although adult Adelie penguins had predominantly LDH-l and LDH-2 in the heart, and LDH-4 and LDH-5 in the pectoralis, all isozymes were present 1n both muscle tissues. They speculated that: Tissues of diving animals might require a larger supply of the LDH isozymes at all points in the pattern, from LDH-l to LDH-5, 1n order to meet physiological requirements under aerobic and also under anaerobic conditions. (p. 144) Although they reported tissue isozyme expression only to the chick stage, their findings are comparable to the isozyme ratios found in the muscle tissues of Pigeon Guillemot young and adult birds. Yokoyama et ale (1979) reported four LDH isozymes present in the heart and pectoralis muscles of the Japanese Lesser Horseshoe bat. There was an increase in LDH-l from embryonic through adult stages, until LDH-l and 2 were predominant in both muscle tissues. They conjectured this increase was an adaptation to flying, an activity that places high aerobic ~etabolic demands on the pectoral muscle. Th1S tIY1ng mammal, then, differs from the diving bird with regards to both LDH isozyme distribution in pectoralis muscle tissue and the metabolic demands placed on the muscle. In the Pigeon Guillemot, the rate and timing of the shift in isozyme expression, from a high ratio of LDH-5 in the fledgling to a more even isozyme distribution in the sexually mature adult, is unknown. This shift in ratios could be gradual or sudden, similar to the 1ncrease that occurs in myoglobin concentration. The change may be correlated with fledging--perhaps the young bird can not fledge unless the total LDH activity reaches some minimum threshold of activity. 29 LDH in the adult Pigeon Guillemot heart muscle shows the most prominent substrate (pyruvate) inhibition (Figure 6). The enzyme reaches maximum velocity at a relatively low concentration of pyruvate, then the velocity decreases as the pyruvate concentration increases further. In comparison, the pectoralis LDH reaches Vmax at a higher pyruvate concentration than heart LDH, and the velocity does not decrease as rapidly with increasing pyruvate concentrations as the heart LDH velocity. The consequences of these differences in substrate inhibition are that pectoralis LDH can tolerate high pyruvate concentrations, enabling the tissue to resort to anaerobic metabolism during hypoxia, while heart LDH can not tolerate high pyruvate concentrations, and must rely on aerobic metabolism. This difference 1n substrate inhibition of the two types of LDH in tissues of Pigeon Guillemots is consistent with that reported for purified LDH isozymes from 8 variety of anima13 (Cahn et Al • 1962; Wils0ll "et al., 1963; Dawson et al., 1964; Kaplan, 1964; Pesce et al., 1964; Vesell and Pool, 1966; Kaplan et al., 1968; Storey and Hochachka, 1974; Muller and Baldwin, 1978; Suarez et al., 1986). Michaelis-Menton constant (Km) estimates (the concentration of the substrate, pyruvate, at one-half Vmax) of LDH are typically lower for cardiac muscle than for skeletal muscle (Appendix E). Km estimates for Pigeon Guillemot LDH also follow this pattern, with estimates for heart muscle tissue approximately one-tenth those for pectorali~ muscle tissue, in the fledgling and adult birds. The chick Kms, however, are lower for both tissues than the fledgling and adult, and the chick heart Km is only approximately one-half that of the chick pectoralis. This suggests that not only do the Kms in both tissues increase as the chick matures to fledgling, but the difference in Kms between the muscles also increases during maturation. These changes in Kms correlate with the changing ratio distribution of LDB isozymes in the tissues. Other enzyme kinetic parameters, such as turnover numbers, would be of interest as they describe the rate at which pyruvate is converted to lactate more thoroughly than the Michaelis-Menton constant (Kaplan and Goodfriend, 1964). Summary Skeletal and heart muscle myoglobin concentrations increase as Pigeon Guillemots mature froa fledglings to adults. The myoglobin concentration in the adult tissues is comparable to that of other birds whose diving ability is simiL~r to that of Pigeon Guillewots. "yo~lohin polypeptide expression changes in the pectoralis muscle as the bird matures from chick to fledgling. Total LDH activity in the pectoralis muscle increases as the chick matures to the fledgling stage; then the activity remains high 1n the adult. The LDH activities in skeletal ~scle are comparable to those found 1n some diving ducks, and lower than those for optimal avian divers such as penguins. The ratio of LDH-5 to other isozymes in the pectoralis muscle increases as the chick matures to the fledgling, where the highest ratio of LDH-5 to other isozymes is found. As the fledgling matures to adult, this ratio decreases, as LDH 1-4 increase in activity. The heart LDH activity and expression of LDB isozymes does not change between the three stages. 30 ~ I I I These findings suggest that the enhancement of myoglobin concentration and the glycolytic enzyme, LDH, and the increase in the ratio of LDH-5 isozyme to other isozymes, enable Pigeon Guillemots to become better metabolically adapted to exercise and diving, as they mature from chicks to adults. The heart and pectoralis muscles in the adults primarily rely on aerobic metabolism, but the pectoralis muscles can potentially resort to anaerobic metabolism during temporary periods of hypoxia. 31 60 ~O 400 -VI E 300c '- 01 c: - 200· t- ::J: