Temperature-dependent modification of divalent cation flux in the rat parotid gland basolateral membrane |
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Authors: | T P Lockwich I H Kim I S Ambudkar |
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Institution: | (1) Secretory Physiology Section, Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Building 10, Room IN-113, 20892 Bethesda, Maryland |
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Abstract: | Divalent cation (Mn2+, Ca2+) entry into rat parotid acinar cells is stimulated by the release of Ca2+ from the internal agonist-sensitive Ca2+ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn2+ influx into internal Ca2+ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca2+-free medium for 10 min) and passive 45Ca2+ influx in basolateral membrane vesicles (BLMV). Mn2+ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn2+ entry appeared to be inactivated since it was not increased by raising extracellular Mn2+] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn2+ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q10=1.7) at 21–37°C to 25 kcal/mol (Q10=3.0) at 21-8°C. Under the same conditions, Mn2+ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E
a
of 7.8 kcal/mol (Q10=1.5) and 6.2 kcal/mol (Q10 < 1.5), respectively. These data suggest that depletion-activated Mn2+ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn2+ entry into unstimulated acini.As in intact acini, Ca2+ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca2+ influx in BLMV at 37°C demonstrated the presence of two Ca2+ influx components: a saturable component, with K
Ca
=279 ± 43 m, Vmax = 3.38 ± 0.4 nmol Ca2+/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but Vmax decreased by 61% to 1.3 ± 0.4 nmol Ca2+/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1 100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca2+ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca2+ uptake at 37°C there was no significant loss of Ca2+ influx. These data suggest that the temperature sensitive high affinity Ca2+ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca2+ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work. |
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Keywords: | Divalent cations Basolateral membrane vesicles Ca2+ flux Mn2+ influx Rat parotid acinar cells Carbachol |
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