Preliminary experiments using squid giant axons show that if these are injected with aequorin and then depolarized with high concentrations of K in seawater, the Ca entry that is measured as an increase in aequorin glow is virtually totally dependent upon Na. These findings suggest two points: (a) that Na/Ca exchange is the principal method whereby Ca enters with depolarization and (b) that the Na/Ca exchange system is electrogenic and hence responds to membrane depolarization by increasing Ca influx and decreasing Ca efflux. A second set of preliminary observations showed squid giant axons are microinjected wit phenol red, and the absorption peaks of this dye are measured in a rotating wheel spectrophotometer, it is possible to make measurements of pH with substantially more sensitivity and better time resolution than can be obtained with a glass microelectrode, which is also used in these measurements. If the squid axon is repetetively stimulated it can be shown that the axoplasm becomes acidified and that upon cessation of stimulation, there is a very slow recovery of pH. The acidification can also be observed if the fiber is depolarized with high [K] seawater and in both instances acidification is totally dependent on the presence of Ca in the seawater. It is concluded that Ca entry which leads to a buffering of the [Ca] also leads to the release of H+ and this phenomenon is to be studied as the means of getting at Ca movement in axoplasm where other methods might not be easy to apply.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS017718-05
Application #
3397793
Study Section
Physiology Study Section (PHY)
Project Start
1981-07-01
Project End
1986-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
5
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Requena, J; Whittembury, J; Scarpa, A et al. (1991) Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin. Ann N Y Acad Sci 639:112-25
Requena, J; Whittembury, J; Mullins, L J (1989) Calcium entry in squid axons during voltage clamp pulses. Cell Calcium 10:413-23
Mullins, L J; Whittembury, J; Requena, J (1989) Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons. Cell Calcium 10:401-12
Requena, J; Mullins, L J; Whittembury, J et al. (1986) Dependence of ionized and total Ca in squid axons on Nao-free or high-Ko conditions. J Gen Physiol 87:143-59
Vassort, G; Whittembury, J; Mullins, L J (1986) Increases in internal Ca2+ and decreases in internal H+ are induced by general anesthetics in squid axons. Biophys J 50:11-9
Mullins, L J; Requena, J; Whittembury, J (1985) Ca2+ entry in squid axons during voltage-clamp pulses is mainly Na+/Ca2+ exchange. Proc Natl Acad Sci U S A 82:1847-51
Requena, J; Whittembury, J; Tiffert, T et al. (1985) The influence of chemical agents on the level of ionized [Ca2+] in squid axons. J Gen Physiol 85:789-804