Effects of parathyroid hormone and N6,O2′-dibutyryl cyclic AMP on Ca2+ transport across the rabbit distal nephron segments perfused in vitro

Effects on Ca²⁺ transport of parathyroid hormone (PTH) and N⁶,O²-dibutyryl adenosine 3,5-cyclic monophosphate (DB-cAMP) were examined in the rabbit distal nephron segments including the cortical thick ascending limb of Henle's loop (CAL), the connecting tubule (CNT) and the cortical collecting tubule (CCT) by the in vitro perfusion technique. When PTH (10^–8 mol·l^–1) was added to the bath, efflux of Ca²⁺ (pmol·mm^–1·min^–1) was increased from 6.29±1.46 to 7.96±1.66 (P<0.02) in the CAL, and from 8.55±1.30 to 13.73±1.24 (P<0.001) in the CNT, respectively, without changes in influx of Ca²⁺. The effect of PTH on Ca²⁺ transport in the CAL, however, was abolished when phosphate concentration in the medium was reduced from 3.0 to 1.0 mmol·l^–1. When DB-cAMP (10^–3 mol·l^–1) was added to the bath, efflux of Ca²⁺ was also increased from 7.01±0.83 to 9.40±0.82, (P<0.05) in the CAL, and from 13.11±0.89 to 19.74±0.52 (P<0.005) in the CNT, respectively. By contrast, neither PTH nor DB-cAMP affected efflux of Ca²⁺ in the CCT. PTH did not affect the transepithelial voltage either in the CAL or in the CNT. But in the CNT, DB-cAMP decreased the voltage from –14.1 to –9.4 mV. The response of adenylate cyclase activity to PTH in the collagenase treated isolated nephron segments was also examined. Significant increases in adenylase cyclase activity were observed in the CAL as well as in the CNT with 10^–6 mol·l^–1 PTH. These data indicate that PTH stimulates Ca²⁺ transport across the CNT probably via activation of the adenylate cyclase-cyclic AMP system. The hormone may also stimulate Ca²⁺ transport across the CAL in a special condition where plasma phosphate concentration is elevated.     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

Summary The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), sprint (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p0.05). In the 51 day E group ATPase activity (0.378±0.009 mol Pi·mg^–1·min^–1) was greater than at 10 and 21 days (0.307±0.006 and 0.323±0.008 mol Pi·mg^–1·min^–1) (p0.05). No change occurred in the S group from 10 to 21 and 51 days (p0.05). Both the 21 and 51 day S (0.318±0.011 and 0.399±0.010 mol Pi·mg^–1·min^–1) and E (0.323±0.008 and 0.378±0.009 mol Pi·mg^–1·min^–1) groups had higher activity compared to the C group (0.193±0.029 and 0.172±0.031 mol Pi·mg^–1·min^–1) (p0.05). Maturation (10–51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca²⁺ binding (p0.05) while Ca²⁺ uptake ability did not change (p0.05). SR yield, Ca²⁺ binding and uptake were not altered with S training (p0.05). The E training resulted in greater Ca²⁺ uptake at 51 days compared to C and S (p0.05), with no change in Ca²⁺ binding (p0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.Supported by grants A-6449 and A-0425 from the Natural Sciences and Engineering Research Council of Canada     

Biphasic short-circuit current response to noradrenaline mediated by Ca2+ and cAMP in cultured rat epididymal epithelium

A study was carried out to investigate the short-circuit current (I _sc) response to noradrenaline (NA) and the signal transduction mechanisms involved in cultured rat cauda epididymal epithelium. In normal Krebs-Henseleit solution, NA (10 mol · l^–1) added basolaterally elicited a biphasic I _sc response consisting of a transient spike followed by a second sustained response. The biphasic response was almost abolished by removing ambient Cl^–. Preloading the tissues witha cell-permeant Ca²⁺ chelator, 1,2-bis(2-aminophenoxy) eth-ane-N,N,N,N,-tetraacetic acid acetoxymethyl ester (BAPTA/AM), or pretreating them with thapsigargin (Tg), a microsomal adenosine triphosphatase inhibitor abolished the initial spike in the I _sc response to NA, but had little effect on the second component. Pretreating the tissues with a non-selective -antagonist, nadolol, reduced the second I _sc response in a dose-dependent fashion but the initial spike was not affected. Microfluorimetric studies showed that NA (100 mol · l^–1) elicited single Ca²⁺ spikes in isolated epididymal cells, which could be abolished by prior treatment with Tg. Biochemical assays showed that NA (10 mol · l^–1) increased intracellular cyclic adenosine monophosphate concentration ([cAMP]_i) and the response was abolished by prior treatment with nadolol (50 mol · l^–1). The results showed that NA elicited a biphasic I _sc response mediated by a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a rise in [cAMP]_i. The Ca²⁺-mediated I _sc response had a faster onset and more transient action than the cAMP counterpart. It is suggested that NA released from noradrenergic nerve endings regulates transepithelial Cl^– secretion in the epididymis thereby providing the specialized millieu vital for sperm storage and maturation.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Comparison of the buffer capacity of endocytotic vesicles,lysosomes and cytoplasm in cells derived from the proximal tubule of the kidney (opossum kidney cells)

During transepithelial acid-base transport cells of the proximal tubule of the kidney have to maintain a relative constant intracellular pH. Herein cellular buffer capacity plays an important role. We measured vesicular buffer capacity in proximal tubule-derived opossum kidney cells and compared it with cytoplasmic buffer capacity to determine the possible importance of vesicular buffer capacity for cellular pH homeostasis. Under HCO₃ ^–-free conditions endocytotic vesicular buffer capacity was 43±4 mmol·l^–1·pH-unit^–1 (n=7) and exceeded cytoplasmic buffer capacity (19±3 mmol·l^–1 ·pH-unit^–1; n=7) significantly. Lysosomal buffer capacity was 19±6 mmol·l^–1·pH-unit^–1; (n=5). Inhibition of vesicular H⁺-ATPase using bafilomycin A₁ led to a dramatic increase of vesicular pH but to a decrease of cytoplasmic pH indicating the importance of organellar buffer systems. We estimated that endocytotic buffer capacity accounts for 23% of cellular buffer capacity under our experimental condition and thus, impairment of endosomal acidification may affect cytoplasmic pH indeed. From our results we conclude that endocytotic vesicles have a large buffer capacity and might play a role in cellular pH homeostasis.     

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig)

The effect of arginine-vasopressin (AVP, 0.1 M) on elementary Ca²⁺ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl₂ as the charge carrier. At a constant potential of –30 mV, bath applied AVP increased the channel openness (NP _o) by a factor of 4.7±3.0 (mean±SD, n=9), the effect resulted from an increase in the frequency of opening (factor 2.5±0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (_o1=0.8±0.4 ms), a double exponential distribution was obtained during AVP (_o1=0.8±0.5 ms, _o2=7.5±0.7 ms). The Ca²⁺ agonist BAYk8644 (1 M) changed the open time distribution similarly to AVP (_o1=1.0±0.5 ms, _o2=9±2.8 ms). With 1 M BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0±1 (n=6). The results are compatible with the idea that AVP can change the gating of L-type Ca²⁺ channels from mode 1 to mode 2.     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at –40 mV associated with a marked increase in conductance. The adrenaline-induced current [I _o (Adr)] reversed polarity near –80 mV. The reversal potential depended distinctly on the external K⁺ concentration but not on external Cl^– concentration. Removal of external Ca²⁺ did not affect I ^o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca²⁺ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid completely abolished I _o(Adr). Furthermore, I _o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an ₁-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I _o(Adr). On the other hand, I _o(Adr) was antagonized by a non-selective -adrenoceptor antagonist phentolamine (0.2 M) and an ₁-adrenoceptor antagonist prazosin (0.2 M), but was not affected by an ₂-adrenoceptor antagonist yohimbine (1 M) or a -adrenoceptor antagonist propranolol (1 M). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca²⁺]_i and an increase in K⁺ conductance. Therefore, it is concluded that adrenaline activates a Ca²⁺-dependent K⁺ conductance by release of Ca²⁺ from internal stores through an activation of an ₁-adrenoceptor.     

Characterisation of Ca2+-dependent inwardly rectifying K+ currents in HeLa cells

The whole-cell configuration of the patch-clamp technique was used to examine K⁺ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10^–7 mol/l free Ca²⁺, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K⁺ by isomolar Na⁺, greatly decreased inward currents and shifted the reversal potential as expected for K⁺ selectivity. The inwardly rectifying K⁺ currents exhibited little or no apparent voltage dependence within the range of from –120 mV to 120 mV. A square-root relationship between chord conductance and [K⁺]₀ at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg²⁺ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K⁺ were: K⁺ (1.0)>Rb⁺ (0.86)>Cs⁺ (0.12)>Li⁺ (0.08)>Na⁺ (0.03). Slope conductance ratios measured at –100 mV were: Rb⁺ (1.66)>K⁺ (1.0)>Na⁺ (0.09)>Li⁺ (0.08)>Cs⁺ (0.06). K⁺ conductance was highly sensitive to intracellular free Ca²⁺ concentration. The relationship between conductance at 0 mV and Ca²⁺ concentration was well described by a Hill expression with a dissociation constant, K _D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba²⁺ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K _D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19 % of the distance across the electrical field of the membrane and a K _D (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mol/l and 3.5 mol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K⁺ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at –120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5–10 mol/l), histamine (1 mmol/l) or bradykinin (1–10 mol/l) to cells dialysed with low-buffered intracellular solutions induced K⁺ currents showing inward rectification and a lack of voltage dependence.     

Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonie epithelial cells (HT-29/B6)

We investigated the inhibitory action of K⁺ channel blockers on carbachol-stimulated Ca²⁺ entry into human Cl^–-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K⁺ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 M) caused a clearly biphasic intracellular calcium (Ca_i response: Ca_i was stimulated from resting levels (85±3 nM, n=100) to a sudden transient peak (821±44 nM) followed by a sustained plateau (317±12 nM). The maintained elevation was dependent on external Ca²⁺ and represented a new steady state between Ca²⁺ entry and exit across the plasma membrane. A monophasic Ca²⁺ response was induced in the absence of external Ca²⁺ and after the initial peak Ca_i returned to baseline. The Ca_i plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 M) or the inorganic Ca²⁺ channel blocker lanthanum (effective concentration for 50% inhibition of Ca₁ plateau EC₅₀=68±18 nM), but it was unaffected by the organic Ca²⁺ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K⁺ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca²⁺ entry. The Ca_i plateau was calculated to be 50% inhibited by barium (96±2 M), lidocaine (74±3 M) and NPPB (27±10 M). The Ca_i plateau was transiently increased by 1 M and 10 M NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl^– channels (so that the membrane potential approached V _K). The resting Ca_i was transiently increased by 50 M and 300 M NPPB to 308±13 nM and 447±153 nM, respectively, suggesting that NPPB induced a Ca²⁺ release from internal Ca stores. We conclude that carbachol-stimulated Ca²⁺ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Ca_i levels to K⁺ channel blockers indicates that there are feedback relationships among rates of Ca²⁺ entry, activity of Ca²⁺-activated K⁺ and Cl^– channels and membrane potential.     

Effect of calcium,furosemide and chlorothiazide on net volume reabsorption and basolateral membrane potential of the distal tubule

In the distal tubule of the isolated kidney of Amphiuma net volume reabsorption (split-oil droplet method) and basolateral membrane potential ( _b ) were measured. Luminal perfusion solution could be changed rapidly from 108 mmol·l^–1 NaCl plus 0.1 mmol·l^–1 calcium to solutions containing 103 or 97 mmol·l^–1 NaCl plus 3.6 or plus 7.2 mmol·l^–1 calcium. Furthermore, 10^–4 mol·l^–1 furosemide or chlorothiazide were applied luminally. (1) Addition of 7.2 mmol·l^–1 calcium hyperpolarized _b from –73.4 mV to –108.3 mV and inhibited net volume reabsorption. (2) Similarly, when furosemide was injected, _b was hyperpolarized and net volume reabsorption reduced. Application of both high calcium and furosemide further inhibited volume reabsorption. (3) The effects of chlorothiazide were similar to those of furosemide. However, when both high calcium and chlorothiazide were administered _b and volume reabsorption were almost normalized. (4) The data are consistent with the hypothesis that calcium and the diuretics interfere primarly with chloride uptake across the luminal membrane and thus reduce sodium chloride transport. When chlorothiazide in the presence of high luminal calcium almost normalized chloride transport, it is likely that its effects were by stimulating calcium transport and thus increasing intracellular calcium activity.Supported by Deutsche Forschungsgemeinschaft (Wi 328)The paper was presented, in part, at the XXVIIth Int. Congr. Physiol. Sci., Paris 1977, Proc. Vol. 13:304     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

ATP and G proteins affect the runup of the Ca2+ current in bovine chromaffin cells

The Ca²⁺ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570±165 pA and then it increased by 49±23% (n=19, SD) over 2±1 min in the absence of adenosine 5-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17±2 min with 2 mM ATP (n=8). Since adenosine 5-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca²⁺ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n=11). Also, guanosine 5-diphosphate (GDP) and guanosine 5-O-(2-thiodiphosphate) (GDP[s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca²⁺ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol. We show that these processes are only effective over a period which parallels the runup and are not additive to it. This suggests that a single process may underlie these various types of facilitation.     

Ca2+ transport in diluting segment of frog kidney

In the isolated-perfused frog (Rana pipiens) kidney the question of whether transepithelial transport of Ca²⁺ is a passive voltage driven process or involves active mechanisms was investigated. With conventional and ion-sensitive microelectrodes transepithelial electrical and electrochemical potential differences were measured. Luminal activities and transepithelial net fluxes of Ca²⁺ and Cl^– were evaluated. Different transepithelial electrical voltages in a wide range (+20 to–4 mV) were generated by chemical voltage clamping and the dependence of Ca²⁺ net fluxes on these voltages investigated. The hormonal control of both Cl^– and Ca²⁺ transport was studied by evaluating the effect of the cell-permeable cAMP analogue, db-cAMP and of the adenylate cyclase stimulator, forskolin. The experiments reveal that: (a) Ca²⁺ is reabsorbed along the diluting segment of frog kidney. (b) Ca²⁺ reabsorption is inhibited by furosemide because of the elimination of the transepithelial voltage. (c) There is a direct relationship between transepithelial voltage and Ca²⁺ reabsorption. (d) Neither Cl^– nor Ca²⁺ reabsorption are affected by db-cAMP or forskolin.We conclude that Ca²⁺ reabsorption is passive, driven by the lumen-positive transepithelial voltage. It most likely occurs via the paracellular shunt pathway.     

Mechanism of hyperthyroidism-induced modulation of the L-type Ca2+ current in guinea pig ventricular myocytes

The positive inotropic effects of thyroid hormone in the heart, increased force and velocity of contraction have been mostly attributed to modulation of myosin ATPase isoenzymes (V₁, V₂ and V₃), and sarcoplasmic reticulum Ca²⁺ pumping activity. In addition, we have suggested that the effects on ventricular contraction result from a thyroid hormone-induced increase in L-type Ca²⁺ current (I _{Ca, L}). Due to the central role of I _{Ca, L} in excitation-contraction coupling, we studied mechanisms whereby thyroid hormone augments this current. Since thyroid hormone modulates adenylate cyclase activity in various tissues, we tested the hypothesis that the hormone activates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A activation, Ca²⁺ channel phosphorylation and increased I _{Ca, L}. We therefore stimulated or inhibited different sites along the adenylate cyclase cascade, and measured I _{Ca, L} and isometric twitch in ventricular myocytes and papillary muscles from euthyroid and hyperthyroid guinea pigs. Our major findings were as follows. In euthyroid myocytes, 0.1 M isoproterenol (Iso) increased I _{Ca, L} (at V _M=0 mV) from –7.04±0.72 to –22.26±1.88 pA/pF, P<0.05, while in hyperthyroid myocytes (I _{Ca, L}=-21.48±2.94 pA/pF), Iso was ineffective. In euthyroid myocytes, intracellular application of cAMP (50 M) was as potent as Iso, but ineffective in hyperthyroid myocytes. In hyperthyroid myocytes, a protein kinase A inhibitor (2 M) lowered I _{Ca, L} from –26.82±1.54 to -10.17±1.70 pApF (P<0.05), but had no effect in euthyroid myocytes. In hyperthyroid myocytes, acetylcholine (ACh) (1 M) decreased I _{Ca, L} from –26.86±1.49 to –18.33±1.25 pA/pF (P<0.05), while in euthyroid myocytes ACh decreased I_{Ca, L} from –6.80±0.61 to –6.00±0.39 pA/pF (NS). Accordingly, in hyperthyroid papillary muscles, ACh decreased twitch tension by 36.4±2.8%, but in euthyroid preparations only by 9.4±5.1% (P<0.05). These findings suggest that thyroid-hormone-induced increase in I _{Ca, L} contributing to positive inotropy, is mediated by activation of the adenylate cyclase cascade.     

Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells

The effects of changing the intracellular concentrations of Ca²⁺ or Mg²⁺ ([Ca²⁺]_i, [Mg²⁺]_i) on Ca current (I _Ca) was studied in frog ventricular myocytes using the whole-cell and cell-attached patch clamp techniques. In the physiological range of [Mg²⁺]_i an increase in [Ca²⁺]_i enhancedI _Ca whereas at lower [Mg²⁺]_i I _Ca was suppressed. The increase inI _Ca caused by Ca²⁺ loading was not mediated by phosphorylation since the kinase inhibitors H-8 {N-[2-(methylamino)-ethyl]-5-isoquinolinesulphonamide dihydrochloride}, staurosporine and KN-62 {1-[N,O-bis(5-isoquinoline-sulphonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine} and a non-hydrolysable adenosine 5-triphosphate analogue ,-methyleneadenosine 5-triphosphate did not prevent the Ca²⁺-inducedI _Ca increase.I _Ca was dramatically increased from 10 ± 6 (n = 4) to 71 ± 7 nA/nF (n = 4) when [Mg²⁺]_i was lowered from 1.0 × 10^–3 to 1.0 × 10^–6 M at a [Ca²⁺]_i of 10^–8 M. The concentration response relation for inhibition of Ca channels by [Mg²⁺]_i is modulated by [Ca²⁺]_i. To account for the experimental results it is postulated that competitive binding of Ca²⁺ or Mg²⁺ to the Ca channel accelerates the transition of the channel from an active to a silent mode. Single-channel recordings support this hypothesis. The regulation may have clinical relevance in cytoprotection during cardiac ischaemia.     

Myofibrillar Ca2+ sensitivity of cardiomyopathic hamster hearts

We studied the Ca²⁺ responsiveness of skinned muscle fibre preparations from the right and left ventricles of normal (FIB) and genetically cardiomyopathic (Bio-To-2) Syrian hamsters. Thus, we compared the Ca²⁺/force relationships of preparations from myopathic hamsters to those of age-matched (11–16 months old) normal animals. The pCa (i.e. –log₁₀[Ca²⁺]) required for 50% force activation (Ca²⁺ sensitivity) was higher in the myopathic hamsters than in controls (pCa₅₀ values of 5.3±0.03 and 5.17±0.04, respectively); this difference might be due to an alteration in regulatory proteins. Indeed, after extraction (with vanadate) and replacement of troponin I with bovine cardiac troponin the pCa₅₀ values were similar (pCa 5.35) to those of bovine ventricular fibres. The Ca²⁺ sensitizer EMD 53998 (10 M) increased Ca²⁺ sensitivity in preparations from normal and cardiomyopathic hamsters equally, by 0.4 pCa units. Incubation of fibre bundles with the catalytic subunit of cyclicadenosine-monophosphate-dependent protein kinase decreased Ca²⁺ sensitivity, thereby normalizing the enhanced Ca²⁺ responsiveness of fibres from cardiomyopathic hamsters. It is not clear, however, whether the pathologically increased Ca²⁺ sensitivity of the hearts of aged myopathic hamsters reflects a maladaptation, or a compensatory mechanism of the failing heart.