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The removal of external Ca2+ ([Ca2+]o) reduces cytosolic Ca2+ ([Ca2+]i) in rat proximal tubules. In this report the role of external Na+ ([Na+]o) on the changes of [Ca2+]i and Ca2+ efflux caused by withdrawal of [Ca2+]o is described in rat renal proximal tubules. In aequorin-loaded tubules [Ca2+]i decreased from 235 +/- 25 to 48 +/- 16 (n = 4, P = 0.017), and 45Ca2+ fractional efflux ratio (45Ca2+ FER) increased from 0.94 +/- 0.03 to 1.64 +/- 0.19 (n = 6, P = 0.021) when Ca2+ was withdrawn from the bathing media of Krebs buffer (KB). The fall of [Ca2+]i, as well as the activation of 45Ca2+ FER, was reversed when [Na+]o in Ca(2+)-free KB was lowered isosmotically from 150 to 15 mM. However, when tubules were superfused with only 5 mM [Na+]o before [Ca2+]o was removed, [Ca2+]i also declined, but 45Ca2+ FER did not increase. The Na(+)-Ca2+ exchange inhibitor dichlorobenzamil (DCB) added after [Ca2+]o was removed evoked responses similar to [Na+]o removal, although DCB also inhibited internal Ca2+ release. These results are congruous with stimulation of Na+ influx in exchange for [Ca2+]i in Ca(2+)-free KB. However, even though total tubular Na+ was higher in Ca(2+)-free KB after 10 min, the initial rate of 22Na+ influx was not different without or with [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)

In the renal proximal tubule, external Ca2+ ([Ca2+]o) is required for parathyroid hormone to elevate cytosolic Ca2+ ([Ca2+]i). However, other hormones increase [Ca2+]i in the absence of [Ca2+]o. These differences may arise from a diversity of signal transduction pathways acting on external and internal Ca2+ pools. However, Ca2+ influx may be necessary to expedite and maintain the rise of [Ca2+]i for a period after the initial surge. In this study, F- was used to probe the roles of intracellular Ca2+ mobilization, Ca2+ influx, and phosphoinositide (PI) hydrolysis on the surge of [Ca2+]i in rat proximal tubules. In the presence of external Ca2+; 1-20 mM F- evoked incremental rises of [Ca2+]i in tubules loaded with aequorin. Whereas 10 mM F- increased [Ca2+]i in the absence of [Ca2+]o, the time constant for the [Ca2+]i surge was increased. These findings are consistent with a role of Ca2+ influx on the effect of F- on [Ca2+]i. Indeed, 10 mM F- also enhanced the uptake of 45Ca2+, and promoted Ca2+ influx in aequorin- and fura-2-loaded, Ca(2+)-deprived tubules. In tubules, F- also activated PI hydrolysis with a time course that paralleled Ca2+ mobilization. The effect of F- on [Ca2+]i was not altered when the 39-kDa pertussis toxin substrate was inactivated with the toxin. This G protein was most likely Gi, because prostaglandin E2, an activator of Gi in tubules, dissociated the pertussis toxin-sensitive protein. The results support the notion that activation of a signal-transduction complex, the F- substrate, causes Ca2+ influx, mobilizes internal Ca2+, and activates PI hydrolysis in rat proximal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

The basolateral cell membrane of the rat proximal tubule contains a Na+-Ca2+ exchanger that may participate in the regulation of cytosolic calcium (Cai) and Ca2+ transport. In this work, the activity and orientation of the Na+-Ca2+ exchanger was studied in rat proximal tubules. The experiments were based on the thermodynamic notion that the exchanger is driven by the prevalence of either of two electrochemical gradients, that for Na+ (delta mu Na+) or for Ca2+ (delta mu Ca2+). Reductions in delta mu Na+, achieved by lowering extracellular Na+ (Nao) from 150 to 15 mM, increased Cai, decreased 45Ca efflux, and increased 45Ca influx. These changes occurred concurrently. When delta mu Na+ was reduced by increasing intracellular Na+ (Nai) with 10(-3) M oubain, Cai also increased. The effect of ouabain was probably dependent on Nai accumulation because the surge in Cai was prevented by exposure of the tubules to 5 mM Nao before ouabain exposure. On the other hand, when delta mu Na+ was lowered mM Nao and then by reducing Nao to 15 mM, Cai rose in two additive stages. We conclude from these data that in the rat proximal tubule the basal state of the Na+-Ca2+ exchanger is in forward mode, Nao-Cai. Moreover, the function of the Na+-Ca2+ exchanger is in accord with predictions derived from a thermodynamic analysis of its function.

Angiotensin-II (AII) and potassium (K+) as stimuli of aldosterone secretion enhance each other's stimulatory potential. In the present study we looked for evidence that AII and K+ act through a common mechanism of signal transduction to affect secretion. Bovine adrenal glomerulosa cells were loaded with the calcium (Ca2+) probe aequorin to permit detection over prolonged time periods of the changes in cytosolic Ca2+ that occur in response to AII and K+. Perfusion fractions were collected for simultaneous measurement of aldosterone production rates. AII (10(-7) M) produced an immediate and transient increase in Ca2+, followed by a Ca2+ plateau that remained above baseline for as long as AII was present. An increase in K+ concentration (from 5 to 12 mM) produced a slow and eventually sustained increase in cytosolic Ca2+, which resembled the plateau produced by AII. Nitrendipine (10(-5) M) completely inhibited the secretory response to AII and K+ (during 60-min incubations) and inhibited the typical K+-induced increase in Ca2+. The sustained increase in Ca2+ with AII (the plateau) required extracellular Ca2+ and was proportional to the prevailing extracellular K+ concentration. When glomerulosa cells were incubated with AII, the aldosterone secretory response to K+ was substantially enhanced (P less than 0.001). In summary, stimulation by both AII and K+ resulted in a sustained increase in Ca2+ influx. AII-induced Ca2+ influx was dependent on the ambient K+ concentration. These results indicate that AII and K+ act together to determine the optimal rate of Ca2+ entry, which may then lead to the appropriate secretory rate of aldosterone.