Possible role of Ca2+-binding sites in the regulation of Na+ transport in toad urinary bladder

La3+ was used to assess the role of membrane-bound Ca2+ in the regulation of basal and antidiuretic hormone (ADH)-induced Na+ transport by the isolated toad urinary bladder. Na+ transport was monitored by means of a short-circuit current (Isc) device. Mucosal La3+ (0.5-5 mM) increased Isc, while serosal La3+ (5 mM) produced a biphasic response (stimulation followed by inhibition). The stimulatory effects of La3+ were additive when present on both sides and were suppressed by mucosal amiloride or serosal ouabain. The action of mucosal La+ was reversible but the inhibition produced by serosal La3+ was not. In the presence of serosal La3+ the natriferic effect of ADH was abolished, but Theophylline, dibutyryl-cAMP, Amphotericin B, mucosal La3+, mucosal low pH, and phospho(enol) pyruvate, were able to increase Isc. These results suggest that Ca2+ binding sites in apical and basolateral membranes may play a key role in the modulation of both basal and ADH-induced Na+ transport. Serosal La3+ apparently inactivates the hormone-receptor interaction and/or the link between the ADH-receptor complex and the activation of adenylate cyclase, but does not interfere with the operation of the Na+ "pump", the basal activity of adenylate cyclase or any of the intracellular events that mediate the effect of ADH on Na+ transport.     

Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium

Summary In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with³H-uridine (30 min pulse–140 min chase), with or without aldosterone (3.5×10^–8 m, 7×10^–8 m) in the presence or absence of SC-9420 (7×10^–6 m, 2.5×10^–5 m) at molar ratios of 2001 to 2801. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5–20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of³H-uridine (30 min pulse–150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change inscc correlated linearly with the fractional change in³H-uridine of 12S cytoplasmic RNA (r=0.95,p<0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of³H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na⁺ transport.     

Amiloride-inhibited Na+ uptake into toad bladder microsomes is Na+-H+ exchange

Amiloride-inhibited Na+ transport into toad urinary bladder microsomes is sensitive to a pH gradient across the vesicular membrane. The magnitude of the gradient was measured directly with acridine orange. Also Na+ could stimulate amiloride-sensitive proton efflux from the microsomes. These results indicated that the transport process was Na+-H+ exchange.     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium.

In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with 3H-uridine (30 min pulse - 140 min chase), with or without aldosterone (3.5 x 10(-8) M, 7 X 10(-8) M) in the presence or absence of SC-9420 (7 X 10(-6) M, 2.5 X 10(-5) M) at molar ratios of 200:1 to 280:1. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5 -- 20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of 3H-uridine (30 min pulse -- 150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change in scc correlated linearly with the fractional change in 3H-uridine of 12S cytoplasmic RNA (r=0.95, p less than 0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of 3H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na+ transport.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

Na+ + K+-ATPase activity and transport processes in toad corneal epithelium

1. Ascorbic acid, diamide and N-ethylmaleimide inhibit Na+ + K+-ATPase activity in toad corneal epithelium. 2. Ascorbic acid, diamide and N-ethylmaleimide increase alpha-aminoisobutyric acid accumulation in this tissue. 3. The effects of these compounds on corneal amino acid and ion transport are not mediated through alterations in Na+ + K+-ATPase activity.     

Extracellular Ca2+ controls outward rectification by apical cation channels in toad urinary bladder: Patch-clamp and whole-bladder studies

Summary Outward rectifying. cation channels were observed in the epithelial cells of the urinary bladder of the toad.Bufo marinus. As studied in isolated cells using the patch-clamp technique, the channel has an average conductance of 24 and 157 pS for pipette potentials between 0 and +60 mV and –60 to –100 mV, respectively, when the major cation in both bath and pipette solutions is K⁺. The conductance of the cannel decreasen with increasing dehydration energy of the permeant monovalent cation in the oder Rb⁺=K⁺>Na⁺>Li⁺. Reversal potentials near zero under biionic conditions imply that the permeabilities for all four of these cations are smiliar. The channel is sensitive to quinidine sulfate but not to amiloride. It shares several pharmacological and biophysical properties with an outwardly-rectifying, vasopressin-sensitive pical K⁺ conductive pathway described previously for the toad urinary bladder. We demonstrate, in both single-channel and whole-bladder studies, that the outward rectification is a consequence of interaction of the chanel with extracellular divalent cations, particularly Ca²⁺, which blocks inward but not outward current. Various divalent cations impart different degrees of outward rectification to the conductive pathway. Concentrations of Mg²⁺ and Ca²⁺ required for halfmaximal effect are 3×10^–4 and 10^–4 m, resopectively. For Co²⁺ the values are 10^–6 m at +50 mV and a 10^–4 m at +200 mV. The mechanism of blockade by divalent cations is not established, but does not seem to involve a voltage-dependent interaction in which the blocker penetrates the transmembrane electric field. In the absence of divalent cations in the mucosal solution, the magnitudes of inward current carried by Rb⁺, K⁺, Na⁺ and Li⁺ through the apical K⁺ pathway at any transepithelial voltage, are in the same order as in the single-channel studies. We propose that the cation channel observed by us in isolated epithelial cells is the single-channel correlate of the vasopressin-sensitive apical K⁺ conductive pathway in the toad urinary bladder and is also related to the oxytocin- and divalent cation-sensitive apical condictivity observed in frog skin and urinary bladder.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

Differential interactions of Na+ channel toxins with T-type Ca2+ channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.     

Temperature dependence of Na+ transport in the isolated toad bladder

• 1.1. Na⁺ transport, measured in the urinary bladder of the toad, was positively dependent on temperature over the range 18–27° and negatively dependent over the range 27–36°. At all temperatures, there was a 1:1 correspondence between radio-isotopically measured net Na⁺ flux and short-circuit current (s.c.c.).
• 2.2. Three possible mechanisms for the positive temperature dependence, i.e. (1) thermal activation of Na⁺ pump, (2) thermal enhancement of metabolism, and (3) thermal facilitation of Na⁺ entry across the apical membrane, were explored by a one-step rapid change in temperature from 18–25° (the temperature-jump method).
• 3.3. In the presence of ouabain, an inhibitor of ATPase, the response to the temperature jump was more impaired than the response to vasopressin, suggesting that thermal activation of the Na⁺ pump may be involved. Metabolic interference with iodoacetate, rotenone or antimycin A abolished the s.c.c. response to the temperature jump, while considerable responsiveness to the stimulating actions of vasopressin and amphotericin B was preserved in the presence of these inhibitors. These results suggest a role for metabolic pathways in mediating thermal activation of Na⁺ transport.
• 4.4. Reduction of the inward Na⁺ gradient significantly reduced the response to the temperature jump, whereas pretreatment with vasopressin or amphotericin B, presumed activators of apical conductance, enhanced the subsequent response to the temperature jump. Thus, heat does not appear to act on the same Na⁺ conductance pathway as do vasopressin or amphotericin B.
• 5.5. These studies indicate that thermal activation of Na⁺ transport does not involve a single unique rate-limiting step, a conclusion consistent with the nonlinear character of the Arrhenius plot.
• 6.6. Owing to the complex character of the temperature dependence of active Na⁺ transport, the response to increments in temperature does not provide useful information on the mechanism of action of mineralocorticoids as implied in an earlier study.

     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells

Stimulus-secretion coupling in pancreatic beta-cells involves membrane depolarization and Ca(2+) entry through voltage-gated L-type Ca(2+) channels, which is one determinant of increases in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca(2+) apparatus further modifies this Ca(2+) signal. When fura-2-loaded mouse beta-cells were depolarized by KCl in the presence of 3 mm glucose, [Ca(2+)](i) increased to a peak in two phases. The second phase of the [Ca(2+)](i) increase was abolished when ER Ca(2+) stores were depleted by thapsigargin. The steady-state [Ca(2+)](i) measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca(2+) pools were depleted. The amount of Ca(2+) presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca(2+)](i) over time (integralDelta[Ca(2+)](i).dt) was approximately 30% higher compared with that in the Ca(2+) pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca(2+)](i) response. Using Sr(2+) in the extracellular medium and exploiting the differences in the fluorescence properties of Ca(2+)- and Sr(2+)-bound fluo-3, we found that the incoming Sr(2+) triggered Ca(2+) release from the ER. Depolarization-induced [Ca(2+)](i) response was not altered by, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca(2+) is not essential for amplification of Ca(2+) signaling. [Ca(2+)](i) response was enhanced when cells were depolarized in the presence of 3 mm glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 microm) diminished the second phase of the depolarization-induced increase in [Ca(2+)](i). We conclude that Ca(2+) entry through L-type voltage-gated Ca(2+) channels triggers Ca(2+) release from the ER and that such a process amplifies depolarization-induced Ca(2+) signaling in beta-cells.