Norio Suda

Modulation of Ca2+ transients and contractile properties by beta-adrenoceptor stimulation in ferret ventricular muscles.

1. The mechanism of modulation of Ca2+ transients and contraction by beta‐adrenoceptor stimulation was studied in ferret ventricular muscles using aequorin to measure intracellular Ca2+. 2. Peaks of tension and light transients were increased by isoprenaline (10(‐9) ‐ 5 x 10(‐7) M) which also abbreviated their time courses. 3. Time‐to‐peak tension was significantly shortened by 5 x 10(‐9) M‐isoprenaline and time‐to‐peak light was abbreviated by 10(‐9) M‐isoprenaline. 4. The time for the light to decay was shortened at 10(‐9) M‐isoprenaline. However, a higher concentration of isoprenaline (10(‐8) M) was required for significant shortening of the half‐relaxation time (TR50). 5. When isoprenaline was removed and beta‐blocker (bupranolol, 1 microM) was applied, the time course of the light transients recovered but the time course of relaxation did not recover. 6. The relationship between [Ca2+]i and tension in tetanic contraction produced in the presence of ryanodine (5 microM) was shifted to the right by isoprenaline (10(‐8) M). This was recovered by the replacement of isoprenaline with bupranolol (1 microM). 7. Isoprenaline (10(‐7) M) added to the solution containing 20 mM [Ca2+]O and Bay K 8644 (1 microM), which produced maximal tension, caused a large light signal and enhancement of the initial phasic tension in tetanic contraction. However, the replacement of isoprenaline with bupranolol after immersing the preparation in 20 mM [Ca2+]O solution with Bay K 8644 and isoprenaline, did not significantly change the tension level, although the light signal decreased. Similar results were obtained in the ventricular muscle of young rats. 8. These results suggest that the dose dependence of modulation of the contractile element and sarcoplasmic reticulum (SR) by beta‐adrenoceptor stimulation differs, and that additional factors, other than the faster Ca2+ uptake by SR and the decrease in Ca2+ sensitivity of the contractile element, might be involved in the shortening of the half‐relaxation time by beta‐adrenoceptor stimulation. In addition, beta‐adrenoceptor stimulation does not produce a marked change in the maximal tension level.

Fluorescence signals from the Mg2+/Ca2+ indicator furaptra in frog skeletal muscle fibers

The fluorescent Mg2+/Ca2+ indicator, furaptra, was injected into single frog skeletal muscle fibers, and the indicators fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, if the value of dissociation constant (KD) for Mg2+ obtained in vitro (5.5 mM) was used. However, if the indicator reacts with Mg2+ with a two-fold larger KD in myoplasm, as previously suggested for the furaptra-Ca2+ reaction (M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor. 1991. J. Gen. Physiol. 97:271-301), the calculated [Mg2+] would average 1.1 mM. Thus, the value 1.1 mM probably represents the best estimate from furaptra of [Mg2+] in resting muscle fibers. Extracellular perfusion of muscle fibers with high Mg2+ concentration solution or low Na+ concentration solution did not cause any detectable changes in the [Mg2+]-related furaptra fluorescence within 4 min. The results suggest that the myoplasmic [Mg2+] is highly regulated near the resting level of 1 mM, and that changes only occur with a very slow time course.

Dihydropyridine‐ and voltage‐sensitive Ca2+ entry in human parathyroid cells

Patch‐clamp and fluorescence measurements of cytoplasmic Ca2+ concentration ([Ca2+]i) were performed to directly detect extracellular Ca2+ entry into cultured parathyroid cells from patients with secondary hyperparathyroidism. Cells loaded with fluo‐3 AM or fluo‐4 AM showed a transient increase in fluorescence (Ca2+ transient) following 10 s exposure to 150 mm K+ solution in the presence of millimolar concentrations of external Ca2+. The Ca2+ transient was completely inactivated after 30–40 s exposure to the high‐K+ solution, was reduced by dihydropyridine antagonists and was enhanced by FPL‐64176, an L‐type Ca2+ channel agonist. The electrophysiological and pharmacological properties of the whole‐cell Ca2+ and Ba2+ currents were similar to those of L‐type Ca2+ channels. The Ca2+ transients induced by 10 s exposure to 3.0 mm extracellular Ca2+ concentration ([Ca2+]o) were inhibited by dihydropyridine antagonists and were partly inactivated following 30–40 s exposure to the high‐K+ solution. These results demonstrate, for the first time, that human parathyroid cells express L‐type‐like Ca2+ channels that are possibly involved in the [Ca2+]o‐induced change in [Ca2+]i. This Ca2+ entry system might provide a compensatory pathway for the negative feedback regulation of parathyroid hormone secretion, especially in hyperplastic conditions in which the Ca2+‐sensing receptor is poorly expressed.

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca2+ indicator dye, fura-2 to investigate the effect of extracellular Ca2+ concentration ([Ca2+]o) on sarcoplasmic reticulum (SR) Ca2+ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca2+]o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca2+ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca2+]o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca2+]o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca2+ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca2+ entry across the sarcolemma during this period. We conclude that in low [Ca2+]o, short stimulation interval may limit Ca2+ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.