Release of 3H-noradrenaline from the rat vas deferens under various in vitro conditions.

The release of 3H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 X 10(-6) M) or low external Na+ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca2+ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca2+ but almost completely in absence of Ca2+. The release by high K+ (117 mM)+low Na+ (26 mM) in normal tissue was dependent on the presence of Ca2+ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca2+ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.     

High spatial resolution studies of muscarinic neuroeffector junctions in mouse isolated vas deferens

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca²⁺ dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and α,β methylene ATP (α,β-MeATP) (1 μM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 μM) elicited Ca²⁺ transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 μM). Ca²⁺ transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 μM). Stimulation produced a marked increase in the frequency and number of Ca²⁺ transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The α₂-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca²⁺ transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional α₂-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca²⁺ events, with no effect on Ca²⁺ transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca²⁺ transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M₃ muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca²⁺ dynamics in this tissue.     

A postsynaptic inhibitory histamine H2-receptor in the mouse isolated vas deferens

The effect of histamine on adrenergic neurotransmission in the mouse isolated vas deferens preparation was investigated. Concentrations of histamine ranging from 0.2 to 650 M depressed, in a dose-related manner, not only the contractile response elicited by field stimulation but also the response caused by the addition of exogenous noradrenaline and acetylcholine. However, the release of [³H]-NA evoked by field stimulation or by high K⁺ remained unchanged in the presence of these concentrations of histamine. The inhibitory effect of histamine on the contractile responses caused by various stimuli was reduced or completely antagonized by cimetidine, a histamine H₂-receptor antagonist but not by mepyramine, a conventional antihistamine. The inhibitory effect of histamine was found to be inversely proportional to both the Ca²⁺ concentration in the bathing medium and to the frequency of field stimulation. Further, the inhibitory effect of histamine was markedly reduced when Mg²⁺ was omitted from the bathing medium. It is concluded that the mouse vas deferens preparation contains a post-junctional inhibitory H₂-receptor. The stimulation of H₂-receptors by histamine inhibits the contractile response of the vas deferens, possibly by decreasing the availability of Ca²⁺ required for contraction by depressing the influx of Ca²⁺.     

Modulation of phospholipase A2 activity in human synovial fluid by cations

Cell-free, Ca²⁺ dependent phospholipase A₂ activity (PLA₂) was measured in human synovial fluid of patients with various kinds of arthritis using [1–¹⁴C] oleate-labelled autoclaved Escherichia coli as substrate. PLA₂ activity at pH 7.0 and with 5 mM added Ca²⁺ was stimulated and then inhibited in a dose-dependent fashion by NaCl; maximal stimulation of 8.8 fold was found at 150 mM Na⁺. Similar effects were obtained with K⁺ Li⁺ and Ru⁺. In the absence of added Na⁺, PLA₂ activity was maximal with 25 mM Ca²⁺ (145 nmols/hr/mg), but in the presence of 150 mM Na⁺, activity was maximal with 4 mM Ca²⁺ (415 nmols/hr/mg). PLA₂ activity was optimal between pH 6.5–8.0 in presence of 150 mM Na⁺¹ and 4 mM Ca²⁺. There was no significant difference between PLA₂ activity in synovial fluids from rhematoid and other types of arthritis. Neutral active, Ca²⁺-dependent PLA₂ activity in acid extracts of human platelets, plasma, polymorphonuclear leukocytes and synovial fluid varied in response to added Na⁺. In presence of 150 mM added Na⁺ and 5 mM PLA₂ activity in human synovial fluid was inhibited by all multivalent cations tested. In the absence of Na⁺, Cu²⁺ and Mg²⁺ stimulated PLA₂ activity in a dose dependent fashion; whereas, Fe²⁺, Fe³⁺ and Al³⁺ were inhibitory. The extent of stimulation by Mg²⁺ was inversely related to the concentration of added Ca²⁺.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Na+/Ca2+ exchange during Ca2+ repletion is not a prerequisite for the Ca2+ paradox in isolated rat hearts

 Ca²⁺ paradox damage has been suggested to be determined by Na⁺ entry during Ca²⁺ depletion and exchange of Na⁺ for Ca²⁺ during Ca²⁺ repletion. Since previously a Ca²⁺ paradox without prior increase of total intracellular [Na⁺] ([Na⁺]_i) has been observed, we investigated whether local accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion plays a role in the Ca²⁺ paradox by replacing all extracellular Na⁺ by Li⁺ 5 min before and during 10 min Ca²⁺-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na⁺- and Ca²⁺-containing solution. Verapamil was used to prevent contracture due to the absence of Na⁺/Ca²⁺ exchange during Na⁺-free perfusion in the presence of Ca²⁺. In group II, the Ca²⁺-free period was omitted, and in group III normal extracellular [Na⁺] was used throughout. ²³Na-NMR was used to monitor intra- and extracellular Na⁺ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca²⁺ paradox in groups I and III. [Na⁺]_i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca²⁺ depletion in group I, whereas in group III [Na⁺]_i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca²⁺-free perfusion. It is concluded that accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion is not a prerequisite for the Ca²⁺ paradox. Received: 2 February 1998 / Received after revision: 31 March 1998 / Accepted: 9 April 1998     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

Characterization of ouabain-induced noradrenaline and acetylcholine release from in situ cardiac autonomic nerve endings

Aim: Although ouabain modulates autonomic nerve ending function, it is uncertain whether ouabain‐induced releasing mechanism differs between in vivo sympathetic and parasympathetic nerve endings. Using cardiac dialysis, we examined how ouabain induces neurotransmitter release from autonomic nerve ending. Methods: Dialysis probe was implanted in left ventricle, and dialysate noradrenaline (NA) or acetylcholine (ACh) levels in the anaesthetized cats were measured as indices of neurotransmitter release from post‐ganglionic autonomic nerve endings. Results: Locally applied ouabain (100 μm ) increased in dialysate NA or ACh levels. The ouabain‐induced increases in NA levels remained unaffected by cardiac sympathetic denervation and tetrodotoxin (Na⁺ channel blocker, TTX), but the ouabain‐induced increases in ACh levels were attenuated by TTX. The ouabain‐induced increases in NA levels were suppressed by pretreatment with desipramine (NA transport blocker) and augmented by reserpine (vesicle NA transport blocker). In contrast, the ouabain‐induced increases in ACh levels remained unaffected by pretreatment with hemicholinium‐3 (choline transport blocker) but suppressed by vesamicol (vesicle ACh transport blocker). The ouabain‐induced increases in NA levels were suppressed by pretreatment with ω‐conotoxin GVIA (N‐type Ca²⁺ channel blocker), verapamil (L‐type Ca²⁺ channel blocker) and TMB‐8 (intracellular Ca²⁺ antagonist). The ouabain‐induced increases in ACh levels were suppressed by pretreatment with ω‐conotoxin MVIIC (P/Q‐type Ca²⁺ channel blocker), and TMB‐8. Conclusions: Ouabain‐induced NA release is attributable to the mechanisms of regional exocytosis and/or carrier‐mediated outward transport of NA, from stored NA vesicle and/or axoplasma, respectively, while the ouabain‐induced ACh release is attributable to the mechanism of exocytosis, which is triggered by regional depolarization. At both sympathetic and parasympathetic nerve endings, the regional exocytosis is because of opening of calcium channels and intracellular calcium mobilization.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Resurgent-like currents in mouse vas deferens myocytes are mediated by NaV1.6 voltage-gated sodium channels

Patch-clamp experiments were performed to investigate the molecular properties of resurgent-like currents in single smooth muscle cells dispersed from mouse vas deferens, utilizing both Na_V1.6-null mice (Na_V1.6^?/?), lacking the expression of the Scn8a Na⁺ channel gene, and their wild-type littermates (Na_V1.6^+/+). Na_V1.6 immunoreactivity was clearly visible in dispersed smooth muscle cells obtained from Na_V1.6^+/+, but not Na_V1.6^?/?, vas deferens. Following a depolarization to +30?mV from a holding potential of ?70?mV (to produce maximal inactivation of the Na⁺ current), repolarization to voltages between ?60 and +20?mV elicited a tetrodotoxin (TTX)-sensitive inward current in Na_V1.6^+/+, but not Na_V1.6^?/?, vas deferens myocytes. The resurgent-like current in Na_V1.6^+/+ vas deferens myocytes peaked at approximately ?20?mV in the current–voltage relationship. The peak amplitude of the resurgent-like current remained at a constant level when the membrane potential was repolarized to ?20?mV following the application of depolarizing rectangular pulses to more positive potentials than +20?mV. 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX), a selective Na_V1.6 blocking toxin, purified from a crude mixture of TTX analogues by LC-FLD techniques, reversibly suppressed the resurgent-like currents. β-Pompilidotoxin, a voltage-gated Na⁺ channel activator, evoked sustained resurgent-like currents in Na_V1.6^+/+ but not Na_V1.6^?/? murine vas deferens myocytes. These results strongly indicate that, primarily, resurgent-like currents are generated as a result of Na_V1.6 channel activity.     

Ionic dependence of electrical activity in small mesenteric arteries of guinea-pigs

The regulation of blood vessel diameter is under the control of the autonomic nervous system (as well as hormones and metabolites), sympathetic nerve stimulation evoking depolarizing post-synaptic potentials. Excitatory junction potentials (EJPs) were recorded from vascular smooth muscle cells of guinea-pig small mesenteric arteries (pressurized) following nerve stimulation. Repetitive stimulation (>5Hz) led to summation of EJPs, which evoked spikes and vasoconstriction. Replacing extracellular Na⁺ with choline (plus atropine) resulted in a decrease in EJP amplitude, but spike amplitude and maximum rate of rise (+V_max) were unaffected. Decreasing the extracellular Ca²⁺ concentration produced decreases in EJP amplitude and spike +V_max, while increasing extracellular Ca²⁺ resulted in increased EJP amplitude and spike +V_max. Verapamil and bepridil, agents that depress Ca²⁺ influx in vascular and visceral smooth muscle, depolarized the membrane and depressed EJPs and spikes at high concentrations (10^–5 M and 5×10^–6 M, respectively). The data indicate that EJPs are dependent on external Na⁺ and Ca²⁺ ions, and that spikes are dependent on Ca²⁺. Thus, neuromuscular transmission in this muscle is similar to that in non-vascular smooth muscles, such as intestinal muscle and vas deferens.Part of this work has been presented to the Biophysical Society (Zelcer and Sperelakis 1980) and to the American Physiological Society (Zelcer and Sperelakis 1981)     

Endogenous and exogenous agonist-induced changes in the coupling between [Ca2+]i and force in rat resistance arteries

The relationship between isometric tension and free cytoplasmic calcium, [Ca²⁺]_i, was investigated in rat isolated resistance arteries using fura-2. Depolarisation with 125 mM K⁺ induced a tonic contraction, while [Ca²⁺]_i increased transiently but stabilised above resting [Ca²⁺]_i. Furthermore, the tension/[Ca²⁺]_i ratio was lower during activation with 125 mM K⁺ if the effect of endogenous noradrenaline (NA) was inhibited. Concentration/ response curves with NA and K⁺ indicated that NA increased the sensitivity to [Ca²⁺]_i. Calcium concentration/response curves in the presence of 10 M NA or 125 mM K⁺ showed that NA could induce force at or below resting [Ca²⁺]_i, while for any given bath calcium concentration, [Ca²⁺]_i was similar in the presence of NA or K⁺. Addition of NA or vasopressin (AVP) to vessels depolarised with 125 mM K⁺ caused force development but no increase in [Ca²⁺]_i, suggesting that agonists increase the efficacy of [Ca²⁺]_i. However, during activation with AVP the efficacy of [Ca²⁺]_i decreased time-dependently. The results suggest that in resistance arteries [Ca²⁺]_i plays a crucial role in excitation-contraction coupling, but the tension/[Ca²⁺]_i relationship can be modified by exogenous and endogenous agonists.     

Ionic regulation of human basophil releasability. II. Non-releasing basophils are converted into releasing basophils in a low-Na+ medium

The effects of different extracellular Na⁺ and CV²⁺ concentrations on histamine release from human basophils were investigated. Isosmotic replacement of extracellular Na ⁺ either with choline ⁺, a non-permeant Na ⁺ analogue, or glucose significantly increased spontaneous and anti-IgE-induced histamine release. Basophils from 12 of 49 normal subjects, which were found not to release histamine upon challenge with an optimal dose of anti-IgE in a 135 mM NaCl buffer, were converted into releasing basophils when stimulation with anti-IgE was performed in a low-Na⁺ medium. The increase in Na ⁺ concentration in the extracellular medium was accompanied by a reduction in the magnitude of basophil response to anti-IgE, which was significantly more pronounced in non-releasers than in releasers (per cent inhibition by 70 mM NaCl 75.5 + 3.2 vs 43.5 + 9.0, P < 0.01). At higher Na⁺ concentrations a progressive and almost complete abrogation of histamine release was observed in non-releasers, but not in releasers (maximal per cent inhibition at 140 mM NaCl 97.3+1.3 vs 50.4 + 8.6). The Na⁺/H⁺ exchanger monensin had a dose-dependent inhibitory effect on anti-IgE-induced histamine release, and the concentration inhibiting 50% of histamine release was l.5 × 10^?7M. When basophils were challenged in the presence of different Na⁺ and C²⁺ concentrations, it was shown that the two cations have antagonistic effects, which is to say that they down-regulate and upregulate histamine release, respectively. Moreover, the requirement of extracellular Ca²⁺ was lowered in a low-Na⁺ medium. These results suggest that Na⁺ and Ca⁺ ions contribute with opposite effects to the modulation of basophil response to anti-IgE and that non-releasing basophils are converted into releasing basophils in a low-Na ⁺ medium.     

Whole-cell currents from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell CCL39

 A conventional patch-clamp technique was used to record the whole-cell current from the cloned canine cardiac Na⁺/Ca²⁺ exchanger NCX1 overexpressed in a fibroblast cell. Ca²⁺ was extracellularly applied to the Na⁺-loaded cell to activate the outward current by operating the reverse mode of NCX1. No measurable outward current was ever elicited from the nontransfected cell. Na⁺/Ca²⁺ exchange blocker 5 mM Ni²⁺ or 3 μM KB-R7943 that was applied extracellularly abolished the outward current. With 140 mM external Li⁺ (replacing Na⁺), the outward current was transient during the Ca²⁺ application. In contrast, with 140 mM external Na⁺, the outward current was maintained without any inactivation during the Ca²⁺ application. I–V relations predicted from the whole-cell clamp protocols used were obtained both before and during the Ca²⁺ application. The exchanger whole-cell currents are thus successfully detectable from NCX1 which is overexpressed in this stable transfectant system. Received: 28 February 1997 / Accepted: 9 April 1997     

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.     

An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction

The hyperpolarization produced by the application of 10^?5 M d-tubocurarine to the end-plate of mouse diaphragm-phrenic nerve preparation pretreated with an irreversible anticholinesterase was studied. This hyperpolarization has been attributed to a non-quantal release of acetylcholine by the nerve terminals. The usual hyperpolarization effect of about 10 mV was mimicked by adding 10^?7 M acetylcholine to the bath which contained 15mM Ca²⁺ to block the non-quantal release. The hyperpolarization effect was maximal (9.2 ± 0.8mV) in saline solution with the normal concentrations of Ca²⁺ (2mM) and K⁺ (5mM). Reducing [Ca²⁺] in the bath decreased the hyperpolarization effect. In Ca-free solution with 4.10^?4 M ethleneglycoltetra-acetate the hyperpolarization effect was3.8 ± 0.6mV. An increase of [Ca²⁺] also reduced the hyperpolarization effect and it was absent in 15mM of Ca²⁺. When the external concentration of Ca²⁺ was kept constant (2mM), both decreasing and increasing K⁺ concentration from 5 mM diminished the hyperpolarization effect. However, the decrease at elevated K⁺ concentrations was due to a reduction of membrane resistance and when the hyperpolarization effect was corrected for the change in size of the miniature endplate potential, it was unchanged in solutions with increased [K⁺] up to 13 mM. No traces of non-quantal release were observed during repetitive stimulation of the phrenic nerve in a medium containing 0.3 mM Ca²⁺. The hyperpolarization effect was not found in denervated muscles that lacked signs of neuromuscular transmission. The hyperpolarization effect was blocked by botulinum toxin while some evoked release of ACh was present. The time required for the block by 1 μg/ml toxin was shorter than for 0.25 μg/ml. The application of α-bungarotoxin produced a hyperpolarization of the membrane in the junctional region. The addition of d-tubocurarine failed to produce the hyperpolarization effect. The hyperpolarization effect was also absent when muscles were incubated with the desensitization potentiating drug, SKF-525A. The hyperpolarization effect was unchanged by replacement of Na⁺ with guanidine and was absent following the replacement of Na⁺ with arginine.The data suggest the channel responsible for the hyperpolarization effect is the same as that giving the endplate potential. The results are in agreement with the hypothesis that the hyperpolarization effect reflects a sustained non-quantal release of acetylcholine from the nerve terminal into the synaptic cleft.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries

The contribution of the Na⁺/Ca²⁺ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (A_SK) with pronounced myogenic tone and mesenteric small arteries (A_MS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na⁺/Ca²⁺ exchanger, the extracellular Na⁺ concentration ([Na⁺]o) was lowered from 143 to 1.2 mM by substituting choline‐Cl for NaCl. The A_SK developed significant myogenic tone and constricted further in low [Na⁺]o. Nifedipine (1 μM ) reduced both myogenic tone and low [Na⁺]o‐induced contraction. Because the membrane potential of A_SK was not changed by low [Na⁺]o (–35 ± 2 mV at 143 mM [Na⁺]o, ?37 ± 3 mV at 1.2 mM [Na⁺]o), depolarization‐induced Ca²⁺ influx was not a cause of the low [Na⁺]o‐induced contraction. The A_MS did not develop significant myogenic tone. Although low [Na⁺]o also constricted A_MS, the magnitude of constriction was significantly weaker than that in A_SK (17 ± 4 vs. 47 ± 6%, P < 0.01, at 58 mM Na⁺). With Bay K 8644, A_MS developed myogenic tone, and low [Na⁺]o‐induced constriction was significantly increased. In conclusion, Na⁺/Ca²⁺ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca²⁺‐extrusion.     

Caffeine stimulates the reverse mode of Na+/Ca2+ exchanger in ferret ventricular muscle

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K‐free medium that blocked the Na⁺–K⁺ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na‐free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca²⁺]_o. Divalent cations Ni²⁺ and Cd²⁺, which have an inhibitory effect on the Na⁺/Ca²⁺ exchanger, produced dose‐dependent inhibition of caffeine responses with apparent Ki of 780 ± 19 and 132 ± 5 μM , respectively. Caffeine also caused dose‐dependent inhibition of Na‐free contractures (Ki=4.62 ± 1.5 mM ), and the reduction or removal of [Na⁺]_o exerted an inhibitory effect on caffeine contractures (Ki=73.5 ± 17.12 mM ). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na⁺/Ca²⁺ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Prostaglandin E2 release from dermis regulates sodium permeability of frog skin epithelium

In the present study we have compared the effects of increased intracellular Ca²⁺ in whole frog skin and isolated epithelium (Rana temporaria). Cellular Ca²⁺ was increased by the use of the endoplasmic reticulum Ca²⁺ ATPase inhibitor, thapsigargin. Serosal addition of thapsigargin to the whole frog skin increased the Na⁺ transport by increasing the apical Na⁺ permeability. This could be blocked by the addition of indomethacin or by removal of Ca²⁺ from the serosal solution. The increase in Na⁺ transport was accompanied by an increased prostaglandin E₂ release. This indicated that the response in Na⁺ transport was due to a Ca²⁺ dependent activation of the prostaglandin E₂ synthesis. Addition of thapsigargin to isolated epithelia inhibited the Na⁺ transport and had no effect on the prostaglandin E₂ release, though the prostaglandin E₂ release from the isolated epithelia could be increased by the addition of arachidonic acid. Addition of prostaglandin E₂ increased the cAMP contents of the isolated epithelia significantly, whereas thapsigargin had no significant effect on the cAMP level. Our results demonstrate that serosal addition of thapsigargin causes a release of prostaglandin E₂ from the dermis below the transporting epithelium. The prostaglandin E₂ diffuses to the epithelium where it activates the Na⁺ transport by increasing cellular cAMP. The epithelium itself does not contribute significantly to the prostaglandin E₂ synthesis. Furthermore an increase in intracellular Ca²⁺ in the epithelial cells without a concomitant increase in prostaglandin E₂ release leads to an inhibition of the active Na⁺-transport.     

Adrenergic and cholinergic inhibition of Ca2+ channels mediated by different GTP-binding proteins in rat sympathetic neurones

Effects of acetylcholine (ACh) and noradrenaline (NA) on voltage-gated ion channels of sympathetic neurones acutely dissociated from rat superior cervical ganglion (SCG) were examined using the whole-cell voltage-clamp technique. Depolarizing voltage steps elicited two types of low- and high-voltage-activated (LVA and HVA) Ca²⁺ currents. Pressure applications of ACh and NA produced concentration-dependent inhibition of the HVA Ca²⁺ current without affecting the LVA Ca²⁺ current. The inhibitory action of ACh on the Ca²⁺ current was blocked by a muscarinic antagonist, atropine. The action of NA was suppressed by an ₂-adrenergic antagonist, yohimbine, but not by an ₁-adrenergic antagonist, prazosin. Delayed rectifying outward K⁺ currents and inward rectifying K⁺ current were not affected by either ACh or NA. Tetrodotoxin-sensitive and -insensitive Na⁺ currents also remained unaffected under actions of ACh and NA. When recorded with electrode containing guanosine-5-O-(3-thiotriphosphate) (GTP--S), the inhibitory actions of ACh and NA on Ca²⁺ currents became irreversible. After treatment of SCG neurones with pertussis toxin, the inhibitory action of ACh on the Ca²⁺ current was almost completely abolished, whereas the action of NA was only partially reduced. The results suggest that ACh and NA differentially inhibit the HVA Ca²⁺ current via different G proteins coupling muscarinic and ₂-adrenergic receptors to Ca²⁺ channels in rat SCG neurones.