The effect of beta-blockade on plasma potassium concentrations and muscle excitability following static exercise

 The effects of β-blockade on plasma [K⁺], muscle excitability and force during fatiguing exercise were examined. Nine healthy males (mean age 22.3±1.7 yr) performed a 3-min fatigue protocol that consisted of a sustained submaximal contraction (30% of the maximal voluntary contraction, MVC) of the right quadriceps muscle. Subjects performed the exercise after treatment with either placebo, β₁-selective (metoprolol, 100 mg) or an equipotent dose of non-selective β_1,2-blockade (propranolol, 80 mg, n=6; 100 mg, n=2; 120 mg, n=1) twice daily for 3 days before testing according to a randomized double–blind design. Brachial arterial and femoral venous blood samples were drawn before, during, and for 15 min following the contraction, together with maximal stimulation of the right femoral nerve to evoke a twitch and a compound muscle action potential (M-wave); the M-wave amplitude being used as an index of sarcolemmal excitability. The exercise-induced rise in plasma [K⁺] did not differ between treatments, but K⁺ re-uptake during recovery was slower following propranolol. The recovery of the twitch was significantly related to the recovery of plasma [K⁺] in all trials, but the evoked M-waves were unaffected by either the contraction or the drug treatment. Propranolol resulted in a significantly (P<0.05) greater reduction (51.9±7.3%) in MVC following the 3-min contraction compared with metoprolol (40.7±3.6%) or placebo (38.9±3.6%). These results suggest that while β_1,2-blockade may significantly affect the recovery of muscle force and K⁺ homeostasis after fatiguing exercise (presumably through an inhibition of the Na⁺,K⁺-ATPase), it does not appear to affect surface membrane excitability. Received: 22 July 1997/Received after revision: 20 January 1998/Accepted: 23 January 1998     

Effect of contraction on lymphatic,venous, and tissue electrolytes and metabolites in rabbit skeletal muscle

Summary The effect of muscle contraction on lymphatic and plasma [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], [P_i], [lactate] ([Lac^–]); [creatine] ([Cr]), ideal osmolality (OSM), and [protein] was evaluated in femoral venous blood and lymph specimens sampled from the calf muscles of rabbits before, in the course of, and after contractions. In addition, total [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], and [H₂O] were analyzed in the muscle tissue. To facilitate lymph sampling both hind limbs were passively flexed and extended, in imitation of natural running movements, by an electrically driven crank. The muscles of one side also performed superimposed rhythmic isotonic contractions. Before contractions, lymphatic [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Lac^–], [Cr], and OSM did not significantly differ from corresponding femoral venous concentrations, [Cl^–], and [P_i] were significantly higher, [protein] significantly lower in the lymph than in the plasma. During contractions lymphatic [K⁺], OSM, [Lac^–], and [P_i] were raised significantly more in the lymph compared with the plasma concentrations. [Na⁺], [Cl^–], [Ca²⁺], and [Mg²⁺] showed only small changes in the course of contractions and thereafter, and they were altered in a similar way in the lymph and plasma. It was suggested that lymphatic and interstitial concentrations were in equilibrium. Comparing inactive with active muscles, the latter lost K⁺ but gained Na⁺, Cl^–, and H₂O, whereas minimal changes occurred in the [Ca²⁺] and [Mg²⁺]. The changes were discussed in connection with the hypothesis that electrolyte shifts might be involved in the activation of the muscular non-proprioceptive interstitial nerve endings which appear to play a role in reflexogenic cardiovascular and respiratory control.A preliminary report of this work has been given elsewhere [33]Supported by Deutsche Forschungsgemeinschaft     

Contribution of pH,diprotonated phosphate and potassium for the reflex increase in blood pressure during handgrip

The relative importance of pH, diprotonated phosphate (H₂PO₄^?) and potassium (K⁺) for the reflex increase in mean arterial pressure (MAP) during exercise was evaluated in seven subjects during rhythmic handgrip at 15 and 30% maximal voluntary contraction (MVC), followed by post-exercise muscle ischaemia (PEMI). During 15% MVC, MAP rose from 92 ± 1 to 103 ± 2 mmHg, [K⁺] from 4.1 ± 0.1 to 5.1 ± 0.1 mmol L^?1, while the intracellular (7.00 ± 0.01 to 6.80 ± 0.06) and venous pH fell (7.39 ± 0.01 to 7.30 ± 0.01) (P < 0.05). The intracellular [H₂PO₄^?] increased 8.4 ± 2 mmol kg^?1 and the venous [H₂PO₄^?] from 0.14 ± 0.01 to 0.16 ± 0.01 mmol L^?1 (P < 0.05). During PEMI, MAP remained elevated along with the intracellular [H₂PO₄^?] as well as a low intracellular and venous pH. However, venous [K⁺] and [H₂PO₄^?] returned to the level at rest. During 30% MVC handgrip, MAP rose to 130 ± 3 mmHg, [K⁺] to 5.8 ± 0.2 mmol L^?1, the intracellular and extracellular [H₂PO₄^?] by 20 ± 5 mmol kg^?1 and to 0.20 ± 0.02 mmol L^?1, respectively, while the intracellular (6.33 ± 0.06) and venous pH fell (7.23 ± 0.02) (P < 0.05). During post-exercise muscle ischaemia all variables remained close to the exercise levels. Analysis of each variable as a predictor of blood pressure indicated that only the intracellular pH and diprotonated phosphate were linked to the reflex elevation of blood pressure during handgrip.     

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A

We identified voltage-activated K⁺ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca²⁺-activated K⁺ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 ± 1.7 pS channels (K_DR1), 90.6 ± 5.4 pS channels (K_DR2) and 149 ± 4 pS intermediate-conductance Ca²⁺-activated K⁺ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from K_DR1 channels inactivated with a time constant of 1.7 ± 0.1 s at +60 mV test potential, while K_DR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca²⁺] (10⁻⁶M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). K_DR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1–10 mM) but not to Ca²⁺. K_DR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of K_DR1 channels 3.4-fold and of K_DR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (−60 mV) only openings of K_DR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K⁺ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells. Received: 20 June 1995/Received after revision: 25 January 1996/Accepted: 7 February 1996     

A transient,RCK4-like K+ current in cultured Xenopus olfactory bulb neurons

 A transient K⁺ current in cultured olfactory bulb neurons of Xenopus tadpoles was studied using the whole-cell patch-clamp technique. The current, which was resistant to 80 mM tetraethylammoniumchloride (TEA) and 10 nM charybdotoxin but blocked by 5 mM 4-aminopyridine (4-AP), activated between −60 and −40 mV and showed time- and voltage-dependent inactivation. Its peak amplitude was nearly independent of the extracellular K⁺ concentration ([K⁺]_o) in the range of 0.05 to 10 mM, indicating that its conductance increased upon increasing [K⁺]_o. The transient K⁺ current showed a slow recovery from inactivation with the time for half-maximum recovery from a conditioning pulse to 80 mV for 1 s varying from 100 ms to 500 ms. Complete recovery required as much as 5–10 s at −80 mV, but could be speeded up at hyperpolarized potentials. The current resembles the RCK4 (Kv1.4) current of rat neurons except that its recovery from inactivation was independent of [K⁺]_o. High-freqency stimulation (20–67 Hz) of the neurons with short (5 ms) voltage pulses resulted in a frequency-dependent, progressive inactivation of the transient K⁺ current. This suggests that, during phasic responses of olfactory bulb neurons, inactivation of the transient K⁺ current occurs and may lead to lengthening of action potentials and facilitation of synaptic transmission. Received: 21 January 1996 / Received after revision: 22 May 1996 / Accepted: 7 June 1996     

Caffeine-evoked contractures in single slow (tonic) muscle fibres of the frog (Rana temporaria and R. esculenta)

Single slow (tonic) muscle fibres were dissected from cruralis muscles of Rana temporaria and R. esculenta. Increasing concentrations of caffeine were applied in Ringer solution, and contractures were measured isometrically. Sigmoid caffeine concentration-response curves were obtained, the threshold value being near 1.2 mmol/l, and maximum contractures being obtained with 10 to 20 mmol/l concentrations of caffeine. Contracture solutions were modified by varying the Ca²⁺ concentration or by replacing Ca²⁺ with 1.8 mmol/l Mg²⁺, Ni²⁺, Co²⁺ or with 0.1–5.0 mmol/l La³⁺. The effects of low pH (5.3), K⁺ (6,10 and 95 mmol/l), adenosine (10 mmol/l) and gallopamil (D600; 30 μmol/l) were examined too. The caffeine threshold was lowered by Mg²⁺, K⁺, 0 .1 mmol/l La³⁺ and D600, while all other substances including 0.5–5.0 mmol/l La³⁺ increased it. The amplitude of contractures evoked by high caffeine concentrations was unaffected. Caffeine (1–40 mmol/l) was also pressure injected into slow fibres. The composition of the solution was modified in a number of ways, but a contractile response was not observed or measured. Extracellular application of caffeine from the same pipettes evoked local contractures. Similar injection experiments in twitch fibres revealed the same results. These observations suggest that an extracellular binding site seems to be involved in the initiation of caffeine-evoked contractures in intact frog muscle fibres. Possible reasons for the ineffectiveness of intracellular caffeine are discussed. Received: 2 September 1995/Received after revision: 22 December 1995/Accepted: 4 January 1996     

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.     

Cardiovascular reflexes during sustained handgrip exercise: role of muscle fibre composition,potassium and lactate

Summary Six healthy men performed sustained static handgrip exercise for 2 min at 40% maximal voluntary contraction followed by a 6-min recovery period. Heart rate (f _c), arterial blood pressures, and forearm blood flow were measured during rest, exercise, and recovery. Potassium ([K⁺]) and lactate concentrations in blood from a deep forearm vein were analysed at rest and during recovery. Mean arterial pressure (MAP) andf _c declined immediately after exercise and had returned to control levels about 2 min into recovery. The time course of the changes in MAP observed during recovery closely paralleled the changes in [K⁺] (r=0.800,P<0.01), whereas the lactate concentration remained elevated throughout the recovery period. The close relationship between MAP and [K⁺] was also confirmed by experiments in which a 3-min arterial occlusion period was applied during recovery to the exercised arm by an upper arm cuff. The arterial occlusion affected MAP whilef _c recovered at almost the same rate as in the control experiment. Muscle biopsies were taken from the brachioradialis muscle and analysed for fibre composition and capillary supply. The MAP at the end of static contraction and the [K⁺] appearing in the effluent blood immediately after contraction were positively correlated to the relative content of fast twitch (% FT) fibres (r=0.886 for MAP vs %FT fibres,P<0.05 andr=0.878 for [K⁺] vs %FT fibres,P<0.05). Capillary to fibre ratio showed an inverse correlation to % FT fibres (r=–0.979,P<0.01). These results indicated that activation of FT rather than slow twitch fibres during static contraction induced a more marked arterial pressure reflex. It was concluded that the arterial pressure reflex would seem to be mediated through stimulation of unmyelinized free nerve endings in the contracted muscle. The [K⁺] would appear to be a more likely candidate than lactate as a mediator for this pressure reflex.     

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells

We have shown previously that secretagogues acting via the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl⁻ conductance, a K⁺ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K⁺ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 μmol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K⁺ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency ( f _c) of 308 ± 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K⁺ selective. 293B reduced the power density of the noise (S _o) to 46 ± 8.7% of its control value and shifted f _c from 291 ± 26 to 468 ± 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5′-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba²⁺ and was completely absent when K⁺ in the bath was replaced by Na⁺. These data suggest that forskolin activates a K⁺ channel of very small conductance which can be inhibited directly and reversibly by 293B. Received: 1 October 1995/Received after revision: 28 December 1995/Accepted: 28 December 1995     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K⁺]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption $\dot V$ O_2peak (30% Rec) and active cycling at 60% $\dot V$ O_2peak (60% Rec). Plasma [K⁺] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints, [K⁺] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol?·?l^?1 to 4.48 (0.19) mmol?·?l^?1 (P?+] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K⁺] increased over the remainder of the recovery periods to 4.36 (0.12) mmol?·?l^?1 in the 30% Rec condition and 4.62 (0.12) mmol?·?l^?1 in the 60% Rec condition, the latter being significantly higher than the former (P?P?P?+] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K⁺].     

Exacerbated potassium-induced paralysis of mouse soleus muscle at 37°C vis-à-vis 25°C: implications for fatigue. K+ -induced paralysis at 37°C

The main aim was to investigate the effects of raised [K⁺]_o on contraction of isolated non-fatigued skeletal muscle at 37°C and 25°C to assess the physiological significance of K⁺ in fatigue. Mouse soleus muscles equilibrated at 25°C had good mechanical stability when temperature was elevated to 37°C. The main findings at 37°C vis-à-vis 25°C were as follows. When [K⁺]_o was raised from 4 to 7?mM, there was greater twitch potentiation, but no significant difference in peak tetanic force. At 10?mM [K⁺]_o there was (1) a faster time course for the decline of peak tetanic force, (2) a greater steady-state depression of twitches and tetani, (3) an increase of peak force over 50?C200?Hz (whereas it decreased at 25°C), (4) significant tetanus restoration when stimulus pulse duration increased from 0.1 to 0.25?ms and (5) greater depolarisation of layer-2 fibres, with no repolarisation of surface fibres. These combined data strengthen the proposal that a large run-down of the K⁺ gradient contributes to severe fatigue at physiological temperatures via depolarisation and impaired sarcolemmal excitability. Moreover, terbutaline, a ??₂-adrenergic agonist, induced a slightly greater and more rapid, but transient, restoration of peak tetanic force at 10?mM [K⁺]_o at 37°C vis-à-vis 25°C. A right shift of the twitch force?Cstimulation strength relationship at 10?mM [K⁺]_o was partially reversed with terbutaline to confer the protective effect. Thus, catecholamines are likely to stimulate the Na⁺?CK⁺ pump more powerfully at 37°C to restore excitability and attenuate, but not prevent, the detrimental effects of K⁺.     

Effects of glucose,forskolin and tolbutamide on membrane potential and insulin secretion in the insulin-secreting cell line INS-1

Membrane voltages (V _m) of INS-1 cells, an insulin-secreting cell line, were measured mostly using the cell-attached mode of the patch-clamp method. The cell-attached configuration allowed the cell to be kept intact. Measurement of V _m was possible because seal resistances were very high and because the membrane obviously had a sufficiently high conductance (probably via K⁺ channels). Resting V _m was −80 ± 1mV (n = 42) and was mainly determined by sulphonylurea-sensitive K⁺ _ATP channels since tolbutamide depolarized the plasma membrane in a concentration-dependent manner and generated action potentials at 50 and 100 μmol/l. D-Glucose, tested between 0.5 and 16.7 mmol/l, also depolarized the plasma membrane in a concentration-dependent manner and induced action potentials at concentrations higher than 5.6 mmol/l. Similarly, forskolin (5 μmol/l) depolarized the cells and increased the frequency of Ca²⁺-mediated action potentials. Insulin secretion was measured from cells growing in culture dishes, by radioimmunoassay. Glucose doubled secretion in INS-1 cells, whereas tolbutamide had no significant effect on secretion in the presence of 0.5 mmol/l and 16.7 mmol/l glucose. At 3 mmol/l glucose, tolbutamide increased insulin release slightly. Forskolin elevated secretion twofold at a low glucose concentration. In contrast, when glucose or tolbutamide were added together with forskolin secretion was potentiated five- to tenfold. These results show that glucose induces membrane activation in INS-1 cells. Furthermore, the potent effect of tolbutamide, i.e. to depolarize the plasma membrane without inducing insulin release, leads to the conclusion that effects distal to depolarization are pivotal for secretion in INS-1 cells. Received: 21 November 1995/Accepted: 18 April 1996     

Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump

 Continued excitation of skeletal muscle may induce a combination of a low extracellular Na⁺ concentration ([Na⁺]_o) and a high extracellular K⁺ concentration ([K⁺]_o) in the T-tubular lumen, which may contribute to fatigue. Here, we examine the role of the Na⁺-K⁺ pump in the maintenance of contractility in isolated rat soleus muscles when the Na⁺, K⁺ gradients have been altered. When [Na⁺]_o is lowered to 25 mM by substituting Na⁺ with choline, tetanic force is decreased to 30% of the control level after 60 min. Subsequent stimulation of the Na⁺-K⁺ pump with insulin or catecholamines induces a decrease in [Na⁺]_i and hyperpolarization. This is associated with a force recovery to 80–90% of the control level which can be abolished by ouabain. This force recovery depends on hyperpolarization and is correlated to the decrease in [Na⁺]_i (r = 0.93; P<0.001). The inhibitory effect of a low [Na⁺]_o on force development is considerably potentiated by increasing [K⁺]_o. Again, stimulation of the Na⁺-K⁺ pump leads to rapid force recovery. The Na⁺-K⁺ pump has a large potential for rapid compensation of the excitation-induced rundown of Na⁺, K⁺ gradients and contributes, via its electrogenic effect, to the membrane potential. We conclude that these actions of the Na⁺-K⁺ pump are essential for the maintenance of excitability and contractile force. Received: 19 December 1996 / Received after revision: 25 March 1997 / Accepted: 2 April 1997     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

K+ as a vasodilator in resting human muscle: implications for exercise hyperaemia

Aim: Potassium (K⁺) released from contracting skeletal muscle is considered a vasodilatory agent. This concept is mainly based on experiments infusing non‐physiological doses of K⁺. The aim of the present study was to investigate the role of K⁺ in blood flow regulation. Methods: We measured leg blood flow (LBF) and arterio‐venous (A‐V) O₂ difference in 13 subjects while infusing K⁺ into the femoral artery at a rate of 0.2, 0.4, 0.6 and 0.8 mmol min^?1. Results: The lowest dose increased the calculated femoral artery plasma K⁺ concentration by approx.1 mmol L^?1. Graded K⁺ infusions increased LBF from 0.39 ± 0.06 to 0.56 ± 0.13, 0.58 ± 0.17, 0.61 ± 0.11 and 0.71 ± 0.17 L min^?1, respectively, whereas the leg A‐V O₂ difference decreased from 74 ± 9 to 60 ± 12, 52 ± 11, 53 ± 9 and 45 ± 7 mL L^?1, respectively (P < 0.05). Mean arterial pressure was unchanged, indicating that the increase in LBF was associated with vasodilatation. The effect of K⁺ was totally inhibited by infusion (27 μmol min^?1) of Ba²⁺, an inhibitor of Kir2.1 channels. Simultaneous infusion of ATP and K⁺ evoked an increase in LBF equalled to the sum of their effects. Conclusions: Physiological infusions of K⁺ induce significant increases in resting LBF, which are completely blunted by inhibition of the Kir2.1 channels. The present findings in resting skeletal muscle suggest that K⁺ released from contracting muscle might be involved in exercise hyperaemia. However, the magnitude of increase in LBF observed with K⁺ infusion suggests that K⁺ only accounts for a limited fraction of the hyperaemic response to exercise.     

Whole-cell conductive properties of rat pancreatic acini

Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a “push-pull” model whereby Cl⁻ is extruded luminally and sequentially taken up basolaterally. In the former model Cl⁻ uptake is coupled to Na⁺ and basolateral K⁺ conductances play a crucial role, in the latter model, Na⁺ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (V _m) more hyperpolarized than −30 mV. In all further studies V _m had to meet this criterion. Under control conditions V _m was −49 ± 1 mV (n = 149). The fractional K⁺ conductance (f _K) was 0.13 ± 0.1 (n = 49). Carbachol (CCH, 0.5 μmol/l) depolarized to −19 ± 1.1 mV (n = 63) and increased the membrane conductance (G _m) by a factor of 2–3. In the seeming absence of Na⁺ [replacement by N-methyl-D-glucamine (NMDG⁺)] V _m hyperpolarized slowly to −59 ± 2 mV (n = 90) and CCH still induced depolarizations to −24 ± 2 mV (n = 34). The hyperpolarization induced by NMDG⁺ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca²⁺. f _K increased to 0.34. The effect of NMDG⁺ on V _m was mimicked by the acidifying agents propionate and acetate (10 mmol/l) added to the bath. The present study suggests that f _K makes a substantial contribution to G _m under control conditions. The NMDG⁺ experiments indicate that the non- selective cation conductance contributes little to V _m in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model. Received: 8 November 1995/Received after revision: 18 December 1995/Accepted: 3 January 1996     

Isokinetic contractile properties of the quadriceps with relation to fiber type

Summary Fifteen subjects were assigned to three groups on the basis of the proportion of fast-twitch fibers (%FT) in their vastus lateralis muscles. Torque production per unit of fat free thigh volume was then determined during knee extension on an isokinetic dynamometer at 60, 120, 180, 240, and 300‡·s⁻¹. Maximal isometric force was also obtained at 65‡ from horizontal. Subjects with predominantly fast twitch muscle fibers demonstrated significantly greater peak power, rate of power production and work than subjects with predominantly slow twitch fibers at all but the lowest velocity of 60‡·s⁻¹. Mean peak power for all subjects occurred at approximately 30% of maximal knee extension velocity (210‡·s⁻¹) and 34% of maximal voluntary isometric contraction. When work, peak power and rate of power production were correlated with %FT fibers, then the highest correlations of 0.69, 0.57, and 0.73 respectively, all occurred at 180‡·⁻¹. These observations suggest that: 1) the torque-velocity, power-velocity relationship of the quadriceps is similar to the force-velocity, power-velocity relationship found for excised muscle, 2) when using the knee extension exercise for the prediction of muscle fiber composition of the quadriceps the most appropriate speed for testing appears to be 180‡·s⁻¹. Supported by a grant from Lumex Inc.     

Block of BK (maxi K) channels of rat pituitary melanotrophs by Na+ and other alkali metal ions

The block of large-conductance calcium-activated potassium (BK) channels by internal and external alkali metal ions was studied in adult rat melanotrophs. Internal but not external 20 mM Na⁺ produced a strongly voltage-dependent, flickery block that was well-fitted to the Woodhull model by using a value of 140 mM for the dissociation rate constant at 0 mV [K _d(0)] and an equivalent valence (zδ) of 0.9. At a concentration of 20 mM external K⁺, Cs⁺ and Rb⁺, but not Li⁺, caused a rightward shift of the voltage dependence of the intracellular Na⁺ (Na⁺ _i ) block. This effect of K⁺, Cs⁺ and Rb⁺ was modelled by an equilibrium knock-out mechanism in which the block-relieving ion binds to a site located within the voltage field and consequently increases the off-rate of Na⁺. Internal Li⁺ caused little or no block whereas internal Cs⁺ caused a voltage-dependent block [K _d(0) ≈150 mM]. Flickery channel block observed in cell-attached patches was consistent with a cytoplasmic Na⁺ activity between 1 and 10 mM. Received: 22 January 1996 /Accepted: 26 March 1996