Jeremy S. Wasser

Effects of dry season dormancy on oxygen uptake, heart rate, and blood pressures in the toad, Bufo paracnemis

The cardiodynamic consequences of dry season dormancy in ectothermic vertebrates is not well known. Our hypothesis was that dormancy would reduce cardiac activity. We therefore determined oxygen uptake and cardiovascular function in aestivating toads, Bufo paracnemis, native to São Paulo State, Brazil. Specimens were collected and kept in the laboratory under controlled temperature and light regimes. We compared oxygen uptake, heart rate, blood pressure, rate-pressure product (RPP), and blood gases in toads during aestivation (dry winter season) and their early active season (spring). Oxygen uptake of winter toads at 25 degrees C was considerably lower than that of spring toads (winter: 24.0 +/- 1.8 ml/(kgh); early spring: 44.4 +/- 5.1 ml/(kgh); mean +/- SE; same in the following). A seasonal dichotomy was also observed at 15 degrees C although the differences was less pronounced (15.8 +/- 1.8 ml/(kgh) winter; 23 +/- 2.1 ml/(kgh) early spring). Chronic arterial cannulation permitted measurements of cardiodynamic variables without any undesired change in VO2. Heart rates of winter toads were significantly lower than those of early spring animals at both experimental temperatures (25 degrees C: winter 25 +/- 1.4 beats/min.; early spring: 35.2 +/- 5.1 beats/min. 15 degrees C: winter 15, 4 +/- 1.8 beats/min.; early spring: 23.9 +/- 2.1 beats/min). Systemic, diastolic and mean arterial pressures decreased slightly but not significantly during aestivation. We conclude that: (1) Bufo paracnemis downregulates metabolic rate during the dry season and (2) heart rate is also downregulated with little change of blood pressure. While the energetics of these responses are probably beneficial for survival during aestivation, the underlying biochemical mechanisms remain obscure.

In vitro tolerance to anoxia and ischemia in isolated hearts from hypoxia sensitive and hypoxia tolerant turtles

Although freshwater turtles as a group are highly anoxia tolerant, dramatic interspecific differences in the degree of anoxia tolerance have been demonstrated in vivo. Painted turtles (Chrysemys picta bellii) appear to be the most hypoxia-tolerant species thus far studied, while softshelled turtles (Trionyx spinifer) are the most hypoxia-sensitive. We have assumed that this dichotomy persists in vitro but have not, until now, directly tested this assumption. We therefore, directly compared the responses of isolated, perfused, working hearts from these two species to either 240 min of anoxia, 90 min of global ischemia, or 240 min of global ischemia followed by reoxygenation/reperfusion. Isolated hearts were perfused at 20 degrees C and monitored continuously for phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi), and intracellular pH (pHi) by 31P-nuclear magnetic resonance spectroscopy as well as for ventricular developed pressure and heart rate. Contrary to our expectations, we observed few significant differences in any of these parameters between painted and softshelled turtle hearts. Hearts from both species tolerated 240 min of anoxia equally well and both restored PCr, pHi, and Pi contents to control levels during reoxygenation. We did observe some significant interspecific differences in the 90 min (pHi and Pi) and 240 min (PCr) ischemia protocols although these seemed to suggest that Trionyx hearts might be more tolerant to these stresses than Chrysemys hearts. We conclude that: (a) the observed in vivo differences in anoxia tolerance between painted and softshelled turtles must either be due to differences in organ metabolism in organs other than the heart (e.g., brain) or to some integrative physiologic differences between the species; and (b) isolated hearts from a species known to be relatively anoxia sensitive in vivo can exhibit an apparent high degree of anoxia and ischemia tolerance in vitro.

31P-NMR determinations of cytosolic phosphodiesters in turtle hearts

As part of our ongoing research on cardiac hypoxia tolerance we have conducted 31P nuclear magnetic resonance (NMR) studies of isolated, perfused, working hearts from freshwater turtles, animals that are well known for their ability to tolerate prolonged periods of anoxia. A striking feature of turtle heart spectra is an extremely high concentration of NMR visible phosphodiesters (PDEs). Cardiac spectra from mammals, on the other hand, typically exhibit only a small resonance in the PDE region. Our aim in this study was to compare myocardial PDE profiles between the highly hypoxia tolerant western painted turtle (Chrysemys picta bellii) and the relatively hypoxia sensitive softshelled turtle (Trionyx spinifer) in order to begin to rest the hypothesis that high constitutive levels of cytosolic PDEs may play a role in conferring hypoxia and ischemia tolerance on the myocardium. We also collected 31P-NMR spectra of PCA extracts of tissue from these species and from Kemps ridley sea turtles (Lepidochelys kempi), as well as spectra from isolated hearts and PCA extracts of red-eared sliders (Trachemys [formerly Pseudemys] scripta]). Total NMR visible phosphodiesters make up 24 +/- 8.6% of the total NMR visible phosphorus in Chrysemys hearts, 20.7 +/- 5.9% in Trachemys hearts, but only 12.2 +/- 5.1% in Trionyx hearts (P < 0.05). We have identified three distinct PDEs in turtle hearts: glycerophosphorylcholine (GPC); glycerophosphorylethanolamine (GPE); and serine ethanolamine phosphodiester (SEP). SEP is the dominant compound in Chrysemys and Trachemys (79.3 +/- 10.2% and 84.7 +/- 3.7% of total PDE, respectively), while GPC is most abundant in Trionyx (74.0 +/- 4.3% of total PDE) and Lepidochelys (not quantitated). The function of this class of compounds is unclear but it has been suggested that cytosolic PDEs may function as lysophospholipase inhibitors, a role that would decrease the rate of membrane phospholipid turnover. Our comparative data suggest that cytosolic PDEs could play a role in phospholipid sparing during anoxic or ischemic stress in turtles but a direct test of this hypothesis awaits future experimentation.

An inductively coupled, doubly tuned resonator for in vivo nuclear magnetic resonance spectroscopy

We present a coil designed for in vivo 31P and 1H nuclear magnetic resonance spectroscopy which consists of a doubly tuned resonator inductively coupled to separate 1H and 31P feed coils. The advantages of the resonator include the ability to 1H shim over the same volume from which 31P spectra are extracted by using a single sample coil, elimination of coupling problems between separate 1H and 31P coils, ease of design and tuning over conventional double-tuned coils, and reduced match/tune sensitivity to coil loading, which is important in in vivo applications. We have used this coil to collect phosphorus spectra from the in situ heart of the western painted turtle (Chrysemys picta bellii) at 2 T. The total heart volume was less than 1 mL and acquisition time was just under 10 min.

Simultaneous optical and nuclear magnetic resonance spectroscopy for monitoring cardiac energetics in vivo

There are a number of applications in which it is useful to simultaneously collect data from what are traditionally separate instrumentation modalities. In particular, in vivo physiological investigations in which data from parallel experiments must be correlated would benefit from simultaneous data collection through 1) elimination of subject variability, 2) elimination of treatment variability, and 3) a reduction in the number of animal preparations required. Here we describe the simultaneous collection of fluo-3 optical fluorescence and /sup 31/P nuclear magnetic resonance (NMR) spectra to measure intracellular calcium levels and high-energy phosphate metabolism, respectively, in vivo. This work is part of ongoing research into the profound anoxia tolerance exhibited by the hearts of certain turtle species. An NMR compatible optical fluorescence spectrometer was constructed and tested. In the 31-cm bore of a 2 T superconducting magnet, NMR and optical spectra were collected every 10-15 min from the in situ, in vivo hearts of anesthetized turtle subjects prior to and during one to three hours of anoxia. It was found that while PCr Stores became significantly depleted during anoxia, /spl beta/-adenosine triphosphate (ATP) levels remained within 20% of control values, and intracellular diastolic calcium levels did not vary by more than 10%. The ability to make simultaneous phosphorus and calcium measurements on a single subject is important to understanding the exact relationship between phosphorus energy state and maintenance of calcium homeostasis.