Negative inotropic effects of aldosterone antagonists in isolated human and guinea-pig ventricular heart muscle

Summary The effects of K⁺-canrenoate (Aldactone^® pro inj.) and its metabolite canrenone on isometric force of contraction were measured in isolated guinea-pig and human papillary muscle preparations driven electrically at a frequency of 1 Hz. In guinea-pig hearts both substances exerted a concentration-dependent negative inotropic effect; the IC₅₀ of K⁺-canrenoate and canrenone were 129±22 µmol l^–1 (n=5) and 85±11 µmol l^–1 (n=12), respectively. At the maximally tested concentration canrenone (250 µmol l^–1) and K⁺-canrenoate (1,000 µmol l^–1) reduced force of contraction by 68±4% (n=12) and 83±3% (n=5), respectively. The negative inotropic effects of canrenone and K⁺-canrenoate were not affected by 10 µmol l^–1 atropine. The negative inotropic effect of canrenone was also not affected by 14 µmol l^–1 aldosterone, but canrenone (10 µmol l^–1) diminished the maximal positive inotropic effect of dihydro-ouabain from 554±75% (n=4) to 269±39% (n=4) of the predrug value.In human heart muscles K⁺-canrenoate and canrenone also exerted a concentration-dependent negative inotropic effect. K⁺-canrenoate (1,000 µmol l^–1) and canrenone (250 µmol l^–1) reduced force of contraction by 57±7% (n=8) and 67±2% (n=6), respectively. A positive inotropic effect of both substances was never observed.It is concluded that the improvement of cardiac performance after application of aldosterone antagonists observed in patients cannot be explained by a direct effect on the heart. K⁺-canrenoate and canrenone are devoid of any direct cardiotonic action. Instead, K⁺-canrenoate and canrenone have direct negative inotropic effects at high concentrations.Abbreviations Aldactone^® pro inj Aldactone^® pro injection - g gramm - Hz Hertz - IC₅₀ concentration of drugs which produce 50% inhibition of force of contraction - i.v. intravenous - min minutes - mm millimeter - mm s^–1 millimeter per second - mmol l^–1 millimolar - mg milligramm - mN milli newton - ms millisecond - SC 8109 spironolactone derivative - SEM standard error of the mean - TRIS Tris(hydroxymethyl)-aminomethan - v/v volume per volume - µmol l^–1 micromolar This work was supported by the Deutsche Forschungsgemeinschaft     

Effects of prostanoids on isolated feline cerebral arteries

The roles of extra-and intracellular calcium for the contractile effects of PGF_2α in the feline basilar artery (BA) were investigated. Comparisons were made with contractions induced by K⁺ and noradrenaline (NA). Addition of nifedipine to PGF_2α-or K⁺ (124 mM)-contracted arteries resulted in an incomplete relaxation, whereas NA-contracted vessels were completely relaxed. Incubation of the preparations in a calcium-free medium containing 10^-5 M EGTA for 5–10 min almost abolished contractions induced by K⁺ and NA. In contrast, 63 % of the response to PGF_2α remained after pretreatment of the arteries in a calcium-free solution for 40 min; PGF_2α produced a biphasic contraction in 17 out of 20 preparations consisting of a rapidly developing initial phase followed by a second increase in tension after 1–6 min. The second phase was absent if the EGTA-concentration was increased to 10^-4 M, or if the arteries were pre-treated with nifedipine. After incubation of the arteries in a calcium-free medium for 40–120 min and K⁺-depolarization, re-addition of calcium elicited contractions at lower concentrations in the presence of PGF_2α than in controls. The results suggest that PGF_2α-induced contractions in the feline BA are considerably less dependent on extracellular calcium than contractions evoked by K⁺ or NA. PGF_2α appears to be able to release calcium from two cellular stores, and may also promote calcium influx through the cell membrane.     

Extracellular K+ concentration during electrical stimulation of rat isolated sympathetic ganglia,vagus and optic nerves

Recordings of extracellular potassium concentration ([K⁺]_e) were made in rat isolated sympathetic ganglia, vagus and optic nerves using ion sensitive microelectrodes. Repetitive orthodromic stimulation of ganglia resulted in [K⁺]_e increases of up to 7 mmol/1 above resting level (6 mmol/1), which were followed by post-stimulus undershoots. Activation of vagal A and B fibres did not significantly alter [K⁺]_e but C-fibre activity induced rises of up to 5 mmol/1. Repetitive stimulation of the predominantly mylinated optic nerve resulted in [K⁺]_e rises of up to 2.5 mmol/1. In the ganglion and vagus nerve, application of ouabain (30–1000 μmol/1) led to a raised baseline [K⁺]_e concentration, an increase in the peak achieved during stimulation and a reduced undershoot amplitude. The amplitude of the undershoot in normal solution was shown to be dependent on the duration of the preceding stimulation period as well as the amplitude of the preceding [K⁺]_e rise. In ganglia and vagus nerves, bath application of γ-aminobutyric acid (10–100μmol/1) and carbachol (10–1000 μmol/1) also elevated [K⁺]_e.It is concluded that repetitive activity in rat peripheral and central nerve fibres leads to significant changes in extracellular K⁺ ion-concentration and that the restoration of these levels is strongly dependent on the intact activity of the membrane Na^+/K⁺ pump.     

The role of the L-arginine/nitric oxide pathway for relaxation of the human lower oesophageal sphincter

Smooth muscle specimens were taken from the oesophagogastric junction (OGJ) in patients operated on for gastrointestinal malignancies not involving the OGJ. The smooth muscle bundles of the inner, circular layer of the OGJ were richly innervated by fine nerve fibres staining positively for NADPH diaphorase. The outer longitudinal layer had a markedly lower number of NADPH-diaphorase positive nerve fibres. When the preparations were suspended in organ baths for recording of isometric tension, they developed active tension. Transmural field stimulation (TMS) induced frequency-dependent relaxations, which were abolished by N^G-nitro-L-arginine (L-NNA; 10^-4 M), and were often converted to atropine-sensitive contractions. The effect of L-NNA was concentration-dependent, and the concentration-response curve for L-NNA was shifted to the right by L-arginine pre-incubation. The enantiomer N^G-nitro-D-arginine (10^-4 M) also showed inhibitory actions on the responses to TMS, but significantly less than L-NNA. Relaxant responses to vasoactive intestinal polypeptide (VIP), forskolin, and sodium nitroprusside were unaffected by L-NNA pre-incubation. Exposure to a 124 mM K⁺ solution resulted in a biphasic relaxation of the preparations. This relaxation was not seen in preparations treated with scorpion venom (20/µg mr^-1) or L-NNA (10^-4M). Instead, a contractile response to 124 mM K⁺ solution was found. The results suggest that NANC responses to electrical stimulation of nerves in the human OGJ are mediated by a product generated from L-arginine. Moreover, the inhibitory biphasic response to 124 mM K⁺ is dependent on the release of this mediator, which seems capable of relaxing the muscle independently of changes in K⁺ conductance.     

Evidence that an L-fucose-containing component in the β-cell plasma membrane is involved in the regulation of glucose-induced insulin release

The effect of the L-fucose-selective lectin Ulex Europeus I (UEA I), a blocker of the Na⁺, K⁺, CIF co-transport system in the kidney, was tested on insulin secretion from isolated β-cell-rich pancreatic islets. UEA I at doses from 50 to 100 μg ml^-1 significantly reduced the glucose-induced (20 mmol l^-1) insulin release whereas the basal (3 mmol l^-1) release was unaffected. The inhibitory effect of 100 μg ml^-1 UEA I was completely abolished by 10 mmol 1-^l L-fucose. The data suggest that an I -fucose-containing structure in the β-cell plasma membrane participates in the regulation of glucose-induced insulin release. This structure may be similar to the L-fucose-containing glycoprotein in the kidney tubules that is believed to be the Na⁺, K⁺, Cl^- co-transporter.     

Some effects of the calcium promotor BAY K 8644 on feline cerebral arteries

The proposed calcium promotor BAY K 86(44) contracted feline basilar arteries partially depolarized by 10 mmol X 1(-1) potassium in a concentration-dependent manner (EC50 value: (2.1 +/- 1.2) X 10(-9) mol X 1(-1)). The concentration-response curve for prostaglandin (PG) F2 alpha was displaced to the left after pretreatment with BAY K 86(44). PGF2 alpha induced a biphasic contraction in calcium-free medium as has been described previously. The second PGF2 alpha-induced contraction phase in calcium-free medium was abolished by pretreatment with nifedipine or diltiazem. BAY K 86(44) restored the second phase in arteries pretreated with nifedipine, but not in vessels pretreated with diltiazem. The findings suggest that BAY K 86(44) acts as a promotor of calcium-influx, probably by interaction with the 'dihydropyridine receptor' in the cell membrane, and also provide support for the view that PGF2 alpha releases membrane-bound calcium.     

Evidence that an l-fucose-containing component in the β-cell plasma membrane is involved in the regulation of glucose-induced insulin release

The effect of the l -fucose-selective lectin Ulex Europeus I (UEA I), a blocker of the Na⁺, K⁺, Cl^? co-transport system in the kidney, was tested on insulin secretion from isolated β-cell-rich pancreatic islets. UEA I at doses from 50 to 100 μg ml^?1 significantly reduced the glucose-induced (20 mmol l^?1) insulin release whereas the basal (3 mmol l^?1) release was unaffected. The inhibitory effect of 100 μg ml l^?1 UEA I was completely abolished by 10 mmol l^?1 l -fucose. The data suggest that an l -fucose-containing structure in the β-cell plasma membrane participates in the regulation of glucose-induced insulin release. This structure may be similar to the l -fucose-containing glycoprotein in the kidney tubules that is believed to be the Na⁺, K⁺, Cl^? cotransporter.     

Plasma K+ changes during intense exercise in endurance-trained and sprint-trained subjects

Active muscle releases K⁺, and the plasma K⁺ concentration is consequently raised during exercise. K⁺ is removed by the NaK pump, and training may influence the number of pumps. The plasma K⁺ concentration was therefore studied in five endurance-trained (ET) and six sprint-trained (ST) subjects during and after 1 min of exhausting treadmill running. Non-exhausting bouts of exercise at either lower speed or of shorter duration were also carried out. Blood samples were taken from a catheter in the femoral vein before and at frequent intervals after exercise. The pre-exercise venous plasma [K⁺] was (mean ± SEM) 3.68±0.10mmol l^-1 (ET) and 3.88 ± 0.06 mmol l^-1 (ST). One minute of exhausting exercise was sustained at 5.27 ±0.08 m s^-1 (ET) and 5.59 ± 0.06 m s^-1 (ST) and caused the plasma K⁺ concentration to rise by 4.4 ± 0.3 (ET) and 4.7 ± 0.3 mmol l^-1 (ST; ns) respectively. Three minutes after exercise the K⁺ concentration was 0.48 + 0.08 mmol l^-1 (ST) and 0.50 ± 0.07 mmol l^-1 (ST) below the pre-exercise value. During the following 6 min of recovery, the value was unchanged for the ET subjects, while a 0.32 ± 0.06 mmol l^-1 rise was seen for the ST subjects. Exercise at reduced intensity or of reduced duration resulted in smaller changes in the K⁺ concentration both during exercise and in the post-exercise recovery, and for each subject the lowest post-exercise K⁺ concentration was therefore inversely related to the peak K⁺ concentration during exercise. For a given peak K⁺ concentration, the ST subjects had higher plasma K⁺ concentrations than the ET subjects in the recovery period, suggesting that the two groups of subjects may regulate the K⁺ concentration differently after exercise.     

Rhythmic contractions in isolated small arteries of rat: role of K+ channels and the Na+, K+-pump

Small mesenteric arteries from Wistar rats display rhythmic tension oscillations, associated with oscillations in membrane potential, when stimulated with noradrenaline. The purpose of this study was to investigate the role of potassium conductance and Na⁺, K⁺-pump activity in the generation of these oscillations. The effect on the rhythmic contractions of several agents, interacting with K⁺ channels, was studied. Application of apamin, pinacidil or glibenclamide did not affect the rhythmic activity. Tetraethylam-monium (TEA) increased the frequency of the rhythmic contractions, while application of 4-aminopyridine (4-AP) increased the amplitude by approximately 50%, with no changes in frequency. Ba²⁺, on the other hand, impaired the rhythmic contractions or converted them to irregular oscillations in the presence of functional endothelium, but did not affect oscillations in endothelium-denuded vessels. Ouabain or exposure to K⁺-free solution, procedures known to inhibit the Na⁺, K⁺-pump, abolished the rhythmic contractions. This effect was immediate, suggesting that it was due to elimination of the electrogenic action of the Na⁺, K⁺-ATPase, rather than to a change in intracellular ion concentrations. Exposure to an extracellular potassium concentration of more than 20 mM also inhibited the oscillation activity. The results suggest that the oscillations are not caused by, but may be modulated by, variations in potassium conductance. The Na⁺, K⁺-pump seems to play an important role in the generation of rhythmic contractions in these vessels.     

Characterization of potassium channels in respiratory cells

In a previous study [26] we described the properties of potassium channels in cultured respiratory cells derived from cystic fibrosis patients (CF) and normal individuals (N). In the present study we examine the regulatory mechanisms of these channels by the patch clamp technique. Since there were no apparent differences in the properties of CF and N K⁺ channels the results were pooled. In the excised inside/out configuration the channel was blocked by different K⁺ channel blockers. Barium (5 · 10^–3 mol/l), retraethylammoniumchloride (5 · 10^–3 mol/l), quinidine 10^–3 mol/l) and lidocaine (5 · 10^–3 mol/l), when added to the cytosolic side, inhibited K⁺ channels reversibly. An increase in the calcium concentration from 10^–7 mol/l to 10^–6 mol/l led to a marked increase in the open channel probability (P _o). Further increases in Ca²⁺ concentration increasedP _o only slightly. No pH effects on the cytosolic side of the channel were observed. The channel open probability was reduced when ATP was present on the cytosolic side at a concentration of 10^–4 mol/l to 10^–3 mol/l. Non hydrolysable adenosine 5-[,-methylene] triphosphate had the same inhibitory effect as ATP. The inhibition by ATP was blunted by the simultaneous addition of 1 mmol/l ADP. The inhibition of K⁺ channels by cytosolic ATP may represent a channel regulatory mechanism in the intact cell. This would allow for coupling between the activity of the (Na⁺+K⁺)-pump and the basolateral K⁺ conductance.     

Changes in extracellular potassium during the spontaneous activity of medullary respiratory neurones

In 34 cats, the changes in extracellular potassium ion activity (a_K) and extracellular spike activity within the pool of respiratory neurones in the dorsormedial and ventrolateral medulla were recorded using microelectrodes filled with a liquid potassium ion exchange resin. Cyclic changes in a_K which parallel central respiratory activity were restricted to those regions where respiratory neurones are known to be localized. The largest changes in a_K (0.1–0.3 mmol · l^–1) were found within the ventral pool of inspiratory neurones. The a_K increased during inspiration in parallel with the pattern of phrenic nerve activity. The smallest changes in a_K (0.02–0.06 mmol · l^–1) were observed within the ventral pool of expiratory neurones. Here, a_K showed a transient increase during both inspiration and expiration. Within the dorsal pool of inspiratory neurones, small fluctuations of a_K were observed paralleling phrenic nerve activity and the afferent discharge of the intact vagal nerves. After the vagal nerves were cut, the changes in a_K then paralleled phrenic nerve activity. The variations in a_K within the ventral pool of respiratory neurones did not change after bilateral section of vagal nerves.Repetitive stimulation of the vagal nerves (0.1–0.5V, 0.05 ms) produced an increase in a_K only within the dorsal pool of inspiratory neurones, whereas repetitive spinal cord stimulation (5–10V, 0.05 ms) resulted in an increase of a_K within the ventral pool of respiratory neurones.The amplitude of the cyclic changes in a_K increased significantly whenever the electrode approached individual respiratory neurones as verified by the amplitude and shape of the spikes recorded by the reference barrel. The maximal changes in a_K then reached a peak amplitude of 1.3–1.5 mmol · l^–1, the pattern of a_K changes resembling that measured within the pools of neurones.The a_K started to rise prior to the discharge of action potentials, indicating that the efflux of K⁺-ions was produced as a consequence of synaptic transmission. The functional importance of these changes in extracellular potassium is discussed.     

Effects of N6, 2‘-0-dibutyryladenosine 3’,5‘-cyclic monophosphate,adenosine, and of oxotremorine and 3-isobutyl-1-methylxanthine on the electrically evoked [3H] acetylcholine secretion in the guinea-pig ileum myenteric plexus

The guinea-pig ileum longitudinal muscle-myenteric plexus preparation, pre-incubated with [³H]choline, was mounted in an organ bath and superfused with Tyrode's solution. [³H]Acetylcholine secretion was evoked by 150 electrical shocks at 0.5 Hz. >N⁶,2′-0-Dibutyryladenosine 3′,5′-cyclic monophosphate (dibutyryl cyclic AMP) enhanced the [³H]acetylcholine secretion in the presence of eserine and the adenosine receptor antagonist 8-phenyltheophylline (10 μmol 1^-1). Conversely, in the absence of 8-phenyltheophylline the [³H]acetylcholine secretion was reduced by dibutyryl cyclic AMP. In the absence and presence of 8-phenyltheophylline (apparent K_D = 12 μ.mol 1^-1), adenosine reduced the [³H]acetylcholine secretion to 33% of control (IC₅₀ = 8, μmol l^-1) and to 48% of control (IC₅₀ = 14 μmol l^-1) respectively. Neither butyrate, dibutyryl cyclic GMP nor guanosine altered the [³H]acetylcholine secretion. Interaction experiments with 3-isobutyl-1-methylxanthine and oxotremorine were done in the absence of eserine, i.e. when oxotremorine is effective. Oxotremorine depressed the fractional secretion of [³H]acetylcholine with a ‘maximal inhibition’ of 13% of control (IC₅₀ = 10 nmol l^-1). In the presence of 3-isobutyl-1-methylxanthine (5 mmol l^-1) oxotremorine depressed the secretion to 2% of control with an apparent IC₅₀) value of 0.9 μmol l^-1 3-Isobutyl-I-methylxanthine (0.01–4 mmol l^-1) enhanced the fractional secretion of [³H]acetylcholine with a ‘maximal enhancement’ value of 232% of control (EC₅₀ = 0.19 mmol l^-1). The presence of oxotremorine (30 nmol l^-1) counteracted, and higher concentrations reversed, the enhancement caused by 3–isobutyl-I-methylxanthine. The results are consistent with the suggestion that the presynaptic muscarinic acetylcholine receptor-mediated control of the secretion of [³H]acetylcholine in the guinea-pig ileum myenteric plexus may be mediated both by cyclic AMP and calcium ions.     

Galanin of the homologous species inhibits insulin secretion in the rat and in the pig

It is known that pig galanin inhibits insulin secretion in dogs, rats and mice. The present study examined whether species-specific, homologous, galanin inhibits insulin secretion. Thus, the effects of rat galanin were examined in the rat, and the effects of pig galanin were examined in the pig, both in vivo and in vitro. In conscious rats, synthetic rat galanin (2 nmol kg^-1) abolished the glucose- (0.56 mmol kg^-1) induced increase in plasma insulin levels. In vitro, rat galanin (10^-9 to 10^-6 mol l^-1) inhibited glucose- (8.3 mmol l^-1) stimulated insulin release from isolated rat islets. In anaesthetized pigs, 15 min infusion of synthetic pig galanin (207 pmol min^-1) into the pancreatic artery decreased the insulin output with a subsequent recovery. In vitro, pig galanin (10^-6 mol l^-1) inhibited glucose- (8.3 mmol l^-1) stimulated insulin release from isolated pig islets. We conclude that homologous galanin inhibits insulin secretion in both the rat and the pig.     

ATP-sensitive K+ channels in rat colonic epithelium

ATP-sensitive K⁺ (K_ATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that K_ATP channels contribute to epithelial K⁺ currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I _sc) induced by the K_ATP channel opener pinacidil (5 10^?4?mol?l^?1) were measured in Ussing chambers under control conditions and in the presence of different blockers of K_ATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K⁺ channel opener, when administered at the serosal side, induced an increase in I _sc consistent with the induction of transepithelial Cl^? secretion after activation of basolateral K⁺ channels, whereas mucosal administration of pinacidil resulted in a negative I _sc. The increase in I _sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10^?4?mol l^?1) and gliclazide (10^?6?mol l^?1), but was resistant even against a high concentration (10^?2?mol l^?1) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I _sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl^? currents across apical Cl^? channels in basolaterally depolarized epithelia indicating that the negative I _sc induced by mucosal pinacidil is due to a transient inhibition of Cl^? secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, K_ATP channels are present in the basolateral membrane of the colonic epithelium; their exact subunit composition, however, has still to be revealed.     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline

 We evaluated the relationship between intracellular calcium concentration ([Ca²⁺]_i) and vasoconstriction during the presence of exogenous noradrenaline (NA) and sympathetic nerve stimulation. An imaging technique was used to determine calcium/tension relationships in isolated rat mesenteric resistance arteries that had been mounted for recording of isometric tension development and loaded with Fura-2/AM. Experiments were performed after depletion of vasodilator neuropeptides and in the continuous presence of 1 μM propranolol, 3 μM indomethacin, and 30 μM nitro-l-arginine. NA (10 μM) was shown first to induce a further increase in tension, but not [Ca²⁺]_i, during the contraction induced by 125 mM K⁺. Subsequently, calcium/tension relationships were determined during stimulation with graded increases in extracellular [K⁺] (5.9–125 mM K⁺), cumulative administration of NA (0.2–10 μM) and electrical field stimulation of perivascular nerves (EFS, 1–16 Hz). A basal calcium/tension relationship without the calcium-sensitizing property of NA was constructed using a cumulative concentration/response curve of 5.9–125 mM K⁺ in arteries after prior exposure to the irreversible α-adrenoceptor antagonist phenoxybenzamine (POB). K⁺ series before and during α-blockade were also studied using the combination of the α₁-antagonist prazosin and α₂-antagonist yohimbine yielding comparable results as with POB. Calcium/tension curves obtained in the presence of NA, K⁺ and during EFS all were shifted to the left compared with the basal condition and all showed a similar slope indicating that neurogenically released NA is equally capable of inducing calcium sensitization in smooth muscle of mesenteric resistance arteries as exogenously applied NA. In the presence of exogenous and endogenous NA we not only observed an elevated contractile response for a given increase in [Ca²⁺]_i, but also an attenuated rise in [Ca²⁺]_i for a given intensity of stimulation. This suggests that the agonist-induced calcium-sensitization is accompanied by a reduction of the rise in [Ca²⁺]_i. Received: 24 October 1997 / Received after revision: 26 January 1998 / Accepted: 26 February 1998     

Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion

The involvement of large-conductance, voltage- and Ca²⁺-activated K⁺ channels (maxi-K⁺ channels) in basolateral Ca²⁺-dependent K⁺-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K⁺ efflux was monitored by measuring the change in K⁺ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na⁺/K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter were inhibited by ouabain (1 mmol/l) and bumeta-nide (50 mol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mol/l) caused a transient large net K⁺ efflux, followed by a smaller K⁺ efflux, which gradually returned to the basal level within 10 min. These two components of the K⁺ efflux appear to be dependent on an increase in cytosolic Ca²⁺ concentration. The initial transient K⁺ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K⁺ efflux transient induced by ACh was inhibited by quinine (0.1–3 mmol/l), quinidine (1–3 mmol/l) and Ba²⁺ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mol/l). Ca²⁺-dependent transient large K⁺ effluxes induced by substance P (0.01 mol/l) and A23187 (3 mol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole-cell outward K⁺ current attributable to maxi-K⁺ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1–10 mmol/l) to the bath. It is concluded that maxi-K⁺ channels are not responsible for the major part of the Ca²⁺-dependent basolateral K⁺ efflux and fluid secretion by the rat submandibular gland.     

Somatostatin inhibition of phospholipase G activity in isolated rat pancreatic islets

We have assessed the effect of somatostatin on the phospholipase C activity in isolated rat pancreatic islets. The phospholipase C activity was measured as the generation of inositol 1,4,5-trisphosphate and its metabolite inositol 1,3,4-trisphosphate from the hydrolysis of polyphosphoinositides. Inositol phosphates were measured using anion-exchange fast protein liquid chromatography analysis of extracts from islets prelabelled with myo-[³H]inositol. Somatostatin (1–1000 nmol l^-1) significantly inhibited the glucose-induced (12 mmol l^-1) phospholipase C activity in a concentration-dependent manner. The Ca²⁺ channel blocker verapamil (25 μmol l^-1) also inhibited the glucose-induced (12 mmol l^-1) phospholipase C, whereas the combination of somatostatin and verapamil did not induce any additional inhibition. At 3.3 mmol l^-1 glucose, the hypoglycaemic sulphonylurea, tolbutamide (1 mmol l^-1), increased the phospholipase C activity. This effect was reversed by somatostatin (100 nmol l^-1). Tolbutamide did not further increase the glucose-induced (12 mmol l^-1) phospholipase C activity. However, the somatostatin inhibition of glucose-induced (12 mmol l^-1) phospholipase C was reversed by tolbutamide. The activator of adenylyl cyclase, forskolin (20 μmol l^-1), did not exert any effect on the PLC-inhibition of somatostatin, whereas forskolin alone inhibited the phospholipase C activation at 12 mmol l^-1 glucose. Our study demonstrates that somatostatin inhibits the hydrolysis of polyphosphoinositides in pancreatic islets, apparently via a mechanism dependent on Ca²⁺ and not on cAMP.     

Effects of dietary sodium on body and muscle potassium content during heat acclimation

Summary It has been suggested that renal conversion of sodium (Na⁺) during training in hot environments results in potassium (K⁺) deficiencies. This investigation examined the influence of two levels of dietary Na⁺ intake (399 vs 98 mmol · d⁻¹) on intramuscular, urinary, sweat, and whole body K⁺ homeostasis. Nine unacclimated, untrained males underwent heat acclimation during two 8 day dietary-exercise regimens (40.1±0.1‡ C, 23.5±0.4%RH). Both diets resulted in depressed urinary K⁺ excretion. Sweat K⁺ and muscle K⁺ concentrations were not altered by diets or acclimation. The whole body stores of Na⁺ increased 31.1% (+ 916.8 mmol) during the high Na⁺ diet and decreased 7.8% (−230.4 mmol) during the low Na⁺ diet; whole body stores of K⁺ increased 4.1% (+ 137.6 mmol) during the high Na⁺ diet and increased 3.4% (+ 113.6 mmol) during the low Na⁺ diet. This dietary-acclimation protocol did not result in whole-body or intramuscular K⁺ deficits and offers no evidence to support previous claims that dietary sodium levels affect K⁺ balance.     

Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma×glioma cells

 By using the whole-cell patch-clamp technique, an inwardly rectifying potassium current, which resembled the ”classic” inward-rectifying potassium current (I _KIR) of other cells in terms of electrophysiological and pharmacological properties, was identified in db-cAMP-differentiated NG108-15 cells. First, the current was dependent on voltage and time. It could be elicited by applying an initial depolarizing prepulse and a subsequent hyperpolarizing command pulse to the cell. The amplitude of the current depended on both the prepulse and the command pulse and increased with the hyperpolarization of the command pulse as well as the depolarization and the prolongation of the prepulse. The activation and inactivation of the current could be fitted well by single-exponential functions and increased with the hyperpolarization of the membrane. Second, the current was dependent on the extracellular potassium concentration ([K⁺]_o). Elevation of [K⁺]_o resulted in a marked increase in the current amplitude and a positive shift of the peak-current/voltage curve as well as the reversal potential. A tenfold increase of [K⁺]_o introduced an ≈43-mV shift of the reversal potential, indicating that the current was carried mainly by K⁺. The conductance (g/g _Max) of the current was also dependent on the [K⁺]_o and increased with increases in [K⁺]_o in a manner approximately proportional to the square-root of [K⁺]_o. Finally, the current was sensitive to Cs⁺ (1 mmol/l), Ba²⁺ (1 mmol/l) and quinidine (0.2 mmol/l); whereas, two typical potassium channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), were weak blockers and reduced the current at high concentration (>10 mmol/l). It was also observed that the current was depressed by Cd²⁺ (1 mmol/l) and Co²⁺ (1 mmol/l) and increased by perfusing the cell with Ca²⁺-free solution. Thus, except for the sensitivity to Cd²⁺, Co²⁺ and Ca²⁺, the current displayed most of the hallmarks described for the ”classic”I _KIR. In conclusion, there appears to be a voltage-dependent I _KIR-type inward rectifier in db-cAMP-differentiated NG108-15 cells. Received: 3 June 1996 / Received after revision: 31 October 1996 / Accepted: 1 November 1996