Calcium Krebs Solution Research Articles

Reduced Antiarrhythmic Efficacy of Verapamil in Isolated Rat Hearts in the Presence of Elevated Extracellular Calcium

An isolated heart method that has been proposed to aid in ascertaining the involvement of L-type calcium channel blockade in the mechanism of action of novel antiarrhythmic drugs involves increasing the calcium concentration in the perfusion buffer. The purpose of this study was to determine the validity of this method using an established L-type calcium channel blocker, verapamil. Isolated rat hearts were perfused with normal calcium (1.4 mM) Krebs solution containing drug vehicle only, a normal calcium solution containing verapamil (300 nM), or a high calcium (2.8 mM) solution containing verapamil. The occurrence of ventricular fibrillation during a subsequent period of regional myocardial ischemia was monitored. The incidence of ventricular fibrillation was significantly reduced from 80% in controls to 20% by perfusion with verapamil in normal calcium Krebs solution (P < 0.05). Perfusion with the high calcium solution increased the incidence of ventricular fibrillation in the presence of verapamil to 40% (P > 0.05 versus controls). We conclude that the antiarrhythmic effect of verapamil in isolated hearts can be attenuated by increasing the calcium content of the perfusion solution, but a twofold increase in the calcium concentration failed to fully restore susceptibility to ventricular fibrillation to that observed in verapamil-free controls.

S-55-2 - Gender differences in the genetic contribution to alcoholism risk: Findings from the Australian twin panel

4-aminopyridine (4-AP, 1 mM) increased noradrenaline (NA) output from the perfused cat spleen at 5 Hz by about fivefold. Enhancement of NA release by 4-AP was reversible. Output of NA induced by potassium (140 mM) was not affected. NA output was doubled at low concentrations (0.1-0.3 mM) of 4-AP, but maximal effect was obtained at 1-3 mM. At 10 mM, it induced spontaneous release of NA which was insensitive to calcium. Insignificant outputs obtained at 5 Hz in 0.1 and 0.3 mM calcium-Krebs solution were markedly enhanced by 4-AP. 4-AP enhanced release at all calcium concentrations up to 5 mM, but maximum output was obtained at 2.5 mM. 4-AP at pH 8.5 was more effective in enhancing NA release than at pH 7.4. In normal spleens the NA output per stimulus at 30 Hz was about four- and eightfold greater than at 5 and 1 Hz, respectively. Reduction of calcium concentration in the Krebs solution to 0.25 mM, while reducing outputs at all frequencies, resulted in an output at 30 Hz, which was nearly ten- and thirtyfold greater than that at 5 and 1 Hz. TEA (10 mM), which markedly enhances NA output, reversed the frequency response so that the maximum output occurred at 1 instead of at 30 Hz. Lowering the calcium concentration to 0.1 mM in the Krebs solution containing TEA, while reducing outputs at all frequencies, increased the output at 30 Hz by about fivefold over the output at 1 Hz. 4-AP, like TEA, enhanced output at lower frequencies (1 and 5 Hz) but did not appreciably affect the output at 30 Hz. The output was always greater at 5 Hz than at 1 Hz. On lowering the calcium concentration to 0.25 mM, the frequency-output relationship became steeper than the controls. 4-AP (1 mM) also enhanced the response to nicotine (50 μM) and it effectively antagonized the suppressant effects of tetrodotoxin (TTX) or calcium-free solution on release induced by nicotine. Lanthanum (1 mM) blocked the restoration of release by 4-AP in calcium-free solution. 4-AP, TEA and guanidine temporarily reversed the inhibition of NA release by guanethidine, an antihypertensive agent, during nerve stimulation or nicotine administration. Since guanethidine does not block conduction of impulses in the preterminal portion of the adrenergic neuron but blocks their propagation in the terminals, the excitation may spread to this portion of the nerve only electrotonically. Since 4-AP, TEA and guanidine are known to mobilize calcium, they may allow greater than normal amounts of calcium to enter the neuron during electrotonic depolarization to restore transmission even in the presence of guanethidine. It is suggested that 4-AP inactivates potassium current in sympathetic nerves and prolongs the duration of the action potential, thereby allowing a greater influx of calcium into the neurone to enhance release of NA. Restoration of nicotine response by 4-AP in calcium-free solution may suggest that 4-AP may mobilize a pool of tightly bound calcium presumably associated with the neuronal membrane. (Supported by USPHS Grant No. HL 22170.)

Non-neurogenic contractions in isolated coronary arteries by brief electrical pulses

The aim was to describe a novel form of non-neurogenic coronary artery contraction. Superfused cattle coronary artery preparations in vitro were placed between platinum electrodes and stimulated. The preparations responded to exceedingly brief transmural stimulation (a single ten thousandth of a second pulse) with long lasting [150(SEM 18.5) s] contractions. These previously undescribed contractions were not reduced by neural blockade nor did they involve free radicals, prostaglandins, or the endothelium. In contrast to the rapid loss of responses to KCl in zero calcium Krebs solution, responses to stimulation were only progressively diminished over many minutes, suggesting involvement of internal calcium stores. The L channel calcium antagonists nifedipine and diltiazem markedly reduced contractions to KCl but did not materially alter those to 1, 5, or 10 stimulation pulses, nor did the T channel antagonist tetramethrin, further supporting the involvement of stored calcium in contractions to stimulation. Evidence was obtained that neither Na+/K(+)-ATPase nor the Na+/Ca+ exchanger are involved in the contractions to stimulation. Ryanodine in zero calcium Krebs solution potentiated contractions to stimulation with 10 pulses and also to endothelin, but depressed contractions to U 46619. Stimulation at excitation parameters well below those associated with neurotransmitter release activates a highly effective contraction sequence that may use a ryanodine insensitive pool of bound calcium. This novel process may have physiological and pathophysiological relevance and provides a means of activating contraction in a coronary artery and studying its time course and contributory components, without the complicating participation of an agonist drug.

Vascular reactivity in the spontaneously hypertensive rat. Effect of high pressure stress and extracellular calcium.

The role of extracellular calcium and high blood pressure stress in altered vascular adrenergic responsiveness in rings of femoral artery from spontaneously hypertensive rats (SHR) was investigated. A model in which partial ligation of the external iliac artery prevents the increase in blood pressure to the ipsilateral femoral artery was used to assess the effect of the increase in pressure stress on these alterations. Age-matched (5-week-old) male Wistar-Kyoto rats (WKY) and SHR were used in the study. Partial ligation was performed before a substantial increase in blood pressure occurred (6 weeks of age), and studies on vascular reactivity were carried out at 10 to 12 weeks of age when the SHR were considered hypertensive (indirect systolic blood pressure greater than 150 mm Hg). Maximal contractility of rings of unprotected femoral artery from the SHR in response to KCl in either a normal (2.5 mM) or low (0.25 mM) calcium Krebs solution was significantly greater (p less than 0.05) than was that of protected vessels from the SHR or protected and unprotected vessels from the WKY; however, no difference in the sensitivity to KCl was observed. Isoproterenol-induced relaxation was significantly attenuated in rings of vascular smooth muscle from unprotected femoral arteries of the SHR (p less than 0.05), while the responses of protected vessels from the SHR were similar to controls. Equilibration of vascular smooth muscle in a low calcium Krebs solution resulted in an increase in beta-adrenergic mediated relaxation in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive inotropic action of amrinone: effect of elevated external Ca2+.

To investigate the positive inotropic effect of amrinone under conditions of maximal contractile stimulation, we compared cumulative amrinone one-half log dose-response curves (dose range, 3.1 X 10(-5)-3.1 X 10(-3) M amrinone) of isolated cat right ventricular papillary muscle in normal (2.5 mM) and high (7.5 mM) calcium Krebs solution. In 2.5 mM calcium, amrinone induced significant (p less than 0.01) dose-dependent increases in peak developed tension (T), maximum dT/dt, and maximum relaxation rate (-dT/dt), which were accompanied by small but significant decreases in time-to-peak tension (TTP). At 10(-3) M, changes in T, dT/dt, and TTP were 61.9 (2.7 g/mm2), 98.1 (33.2 g/s/mm2), and -13.1% (-26 ms), respectively. In 7.5 mM calcium, the increases in T, dT/dt, and -dT/dt, and the reduction of TTP, were markedly less at each concentration. At 10(-3) M amrinone, T and dT/dt increased significantly by 16.7 (1.3 g/mm2) and 23.5% (17.4 g/s/mm2), respectively, with no change in TTP. The amrinone-induced reductions in one-half relaxation time and twitch duration persisted in high calcium and were similar in magnitude to those in 2.5 mM calcium. The substantial reduction of amrinone's positive inotropic effect in high Ca2+ suggests that a major part of the drug's action involves an augmentation of contractile Ca2+. An amrinone-induced effect on rate of Ca2+ delivery to the contractile apparatus or on myofilament Ca2+ sensitivity would be consistent with a persistent inotropic action in high Ca2+. Contractile changes induced by amrinone are discussed in relation to a possible effect on intracellular cyclic AMP levels.

The effect of prostaglandin E2 on histamine-stimulated calcium mobilization as a possible explanation for histamine tachyphylaxis in canine tracheal smooth muscle.

Isolated strips of canine tracheal smooth muscle rapidly lost their responsiveness to histamine when placed in a zero calcium Krebs buffer. Responsiveness to acetylcholine, however, was not rapidly lost, and following 120 min of incubation in zero calcium buffer with frequent washes, 10% of the contractile response still remained. The kinetics of each loss of response suggest that primarily a loosely bound source of calcium is mobilized by histamine and a more tightly bound source is mobilized by acetylcholine. Consistent with these data were the effects of the calcium antagonist verapamil. In normal calcium Krebs solution, dose-response curves to histamine were markedly reduced by verapamil while acetylcholine responses were relatively unaffected. In calcium depleted tracheal strips, indomethacin potentiated the calcium dose-response curve, determined by incremental readdition of calcium in the presence of histamine (10(-4) M), with comparatively little effect on the calcium dose-response curve in the presence of acetylcholine (10(-6) M). Also, in indomethacin pretreated tracheal strips, a reduction in the histamine-calcium dose-response curve could be produced by exogenous addition of 2.8 X 10(-9) M and 2.8 X 10(-8) M PGE2. In the acetylcholine-calcium responses there was a significant reduction only at 2.8 X 10(-8) M PGE2. These data suggest that histamine mobilizes primarily a loosely bound, possibly extracellular source of calcium necessary for contraction, and this histamine-stimulated calcium mobilization is sensitive to the effects of PGE2.

The role of calcium in the effects of noradrenaline and phenoxybenzamine on adrenergic transmitter release from atria: no support for negative feedback of release.

1 The relation of calcium ion influx into nerve terminals to presynaptic adrenoceptor function and the possible masking, by desensitization due to intraneuronal calcium accumulation, of the effects of adrenoceptor agonists and antagonists on presynaptic alpha-adrenoceptors was investigated in guinea-pig atria previously incubated with [(3)H]-noradrenaline.2 Atria were stimulated with 100 pulses at various frequencies (1 to 15 Hz) in standard (2.3 mm), low (0.26 mm) and high (6.9 mm) calcium-Krebs solution in the absence and then the presence first of noradrenaline and subsequently phenoxybenzamine.3 The per pulse overflow of tritium was directly related to the calcium concentration of the Krebs solution, being much reduced and substantially increased in 0.26 and 6.9 mm calcium-Krebs solutions respectively.4 Noradrenaline inhibited the overflow of tritium in low calcium-Krebs solution, to a relatively constant extent, independently of frequency. In addition, the agonist had a greater maximal inhibitory effect in standard than in reduced calcium-Krebs. The catecholamine was as effective an inhibitor of overflow at the lowest and highest frequencies in high as it was in standard calcium-Krebs solution. Phenoxybenzamine invariably increased the tritium overflow but was generally less effective both in low and in high calcium-Krebs solution. The patterns of inhibition and enhancement of stimulation-induced tritium overflow by these two agents do not indicate an intimate relationship between calcium influx and adrenoceptor activation; nor does desensitization appear to be an adequate explanation of the relationship between frequency of stimulation and the intensity of agonist and antagonist effect in the three different calcium concentrations.5 It is concluded that the perineuronal levels of adrenergic transmitter do not establish the magnitudes of effect of exogenous adrenoceptor agonists and antagonists on tritium overflow and that a negative feedback regulation of release by transmitter is exceedingly unlikely under ordinary conditions of neurotransmission.

Effect of guanidine on release of noradrenaline from the perfused spleen of the cat.

1 Guanidine increased noradrenaline (NA) output at 5 Hz by 3 to 6 fold, and doubled it at 30 Hz. Onset of maximum activity was slow, and reversal was also slow. Output of NA induced by potassium, sodium deprivation, or tyramine was not affected. 2 NA output was doubled at low concentrations (1 to 2 mM) of guanidine, but maximal effect was obtained at 4 mM. At 10 mM, spontaneous release was occasionally observed. 3 The effect of guanidine on NA release was related to the external calcium concentration. Outputs which previously have been shown to be insignificant at 5 Hz in 0.25 and 0.75 mM calcium-Krebs solution were markedly enhanced by guanidine. Guanidine enhanced release at all calcium concentrations up to 7.5 mM, but maximum output was obtained at 2.5 mM. 4 Guanidine had no effect on the recovery of intra-arterially infused NA. 5 The effects of guanidine and tetraethyl-ammonium (TEA) on NA release at 5 Hz were additive. 6 Guanidine reversed the inhibition of NA release by guanethidine during nerve stimulation at 5 and 10 Hz, and the NA output increased nearly 2.5 fold after repeated stimulation of the nerves. Guanidine was less effective in reversing the inhibitory effects of guanethidine on NA release at 30 Hz. 7 Guanidine did not affect release of catecholamines (CA) from the perfused cat adrenal gland by splanchnic nerve stimulation. 8 It is suggested that guanidine enhances NA release partly by increasing the influx of calcium into the neurone during an action potential, and also by interfering with intracellular binding of calcium.

Effect of 4-aminopyridine on release of noradrenaline from the perfused cat spleen by nerve stimulation.

1. 4-aminopyridine (4-AP, 1 mM) increased noradrenaline (NA) output from the perfused cat spleen at 5 Hz by about fivefold. Enhancement of NA release by 4-AP was reversible. Output of NA induced by potassium was not affected. 2. NA output was doubled at low concentrations (0.1--0.3 mM) of 4-AP, but maximal effect was obtained at 1--3 mM. At 10 mM, it induced spontaneous release of NA which was insensitive to calcium. 3. Insignificant outputs obtained at 5 Hz in 0.1 and 0.3 mM calcium-Krebs solution were markedly enhanced by 4-AP. 4-AP enhanced release at all calcium concentrations up to 5 mM, but maximum output was obtained at 2.5 mM. 4. 4-AP at pH 8.5 was more effective in enhancing NA release than at pH 7.4. 5. 4-AP increased the recovery of intra-arterially infused NA from the control 26 to 47%. 6. 4-AP did not affect release of catecholamines (CA) from the perfused cat adrenal gland by acetylcholine (ACh). 7. It is suggested that 4-AP inactivates potassium current in sympathetic nerves and prolongs the duration of the action potential, thereby allowing a greater influx of calcium ions into the neurone to enhance release of NA.

Release of noradrenaline from slices of cat spleen by pre-treatment with calcium, strontium and barium.

1. Spleen slices pre-incubated for different periods at 4 degrees C in Krebs solution containing varying concentrations of calcium, up to 96 mM, lost their endogenous noradrenaline stores when reincubated in normal Krebs solution at 37 degrees C for 2 hr. Rate of loss of noradrenaline was roughly related to the calcium concentration of the pre-incubation medium and the pre-exposure time.2. Pre-treatment with isotonic barium or strontium (96 mM) Krebs solution also induced release of noradrenaline from spleen slices when re-exposed to normal Krebs solution. Barium was more effective than either calcium or strontium.3. The enhanced release induced by calcium pre-treatment occurred in the absence of calcium, with or without EGTA.4. Tissue calcium concentration of spleen slices was 0.68 m-mole/kg. Pre-treatment of slices with normal or 96 mM calcium-Krebs solution for 4 hr at 4 degrees C increased the calcium concentration to 2.57 and 9.9 m-mole/kg, respectively.5. Ouabain, which caused a dose-dependent release of noradrenaline, did not modify the release induced by calcium pre-treatment.6. Spleen slices prepared from cats anaesthetized with sodium pentobarbitone instead of ether were resistant to noradrenaline depletion by calcium pre-treatment.7. Evoked release of [(3)H]noradrenaline by high potassium from calcium-pre-treated slices did not occur in the absence of external calcium, even though the calcium pre-treatment enhanced the tissue concentration of this ion by nearly tenfold.8. Net uptake of noradrenaline in normal and in treated slices whose noradrenaline content was severely reduced by barium pre-treatment or sodium withdrawal was comparable.9. Specific activity of released and endogenous [(3)H]noradrenaline increased as the tissue stores of noradrenaline were reduced.10. It is suggested that the spontaneous loss of tissue noradrenaline after pre-treatment with high-calcium solution was due to inhibition of sodium-potassium-activated ATPase by intracellular accumulation of calcium ions. Evidence is presented to suggest that vesicles depleted of their endogenous transmitter by pre-treatment with calcium, strontium or barium, or by sodium withdrawal, are re-used for the storage and release of exogenous noradrenaline.