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Curr Res Physiol
2023 Jan 01;6:100100. doi: 10.1016/j.crphys.2023.100100.
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Long-term Xenopus laevis tadpole -heart-organ-culture: Physiological changes in cholinergic and adrenergic sensitivities of tadpoleheart with thyroxine-treatment.
Hanada H
,
Morishita F
,
Sanoh S
,
Kashiwagi K
,
Kashiwagi A
.
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The present study clarified changes in physiological sensitivities of cultured Nieuwkoop and Faber stage 57 Xenopus laevis tadpole-organ-heart exposed to thyroxine (T4) using acetylcholine (ACh), norepinephrine (NE) and atropine. For preliminary life span and the chemical tests, 60% minimum essential medium (MEM), two types of modified Hank's balanced salt-solution-culture-media (MHBSS-CM) I and II containing relatively lower concentrations of amino acids and collagen were prepared. In preliminary lifespan-test of cultured tadpole hearts, the hearts maintained in 60% MEM was 50 days on average, whereas that of the tadpole-hearts in MHBSS-CMs was extended by 109 days on average, showing superior effectiveness of MHBSS-CMs. 4 min-stimulation by 5 × 10-9 M T4 tended to increase the tadpole heartbeat. 10-9 M ACh decreased the tadpole heartbeat. Frog-heart at 2-4 weeks after metamorphosis completion and tadpoleheart treated with 5 × 10-10 M T4 for 45 h also responded to 10-9 M ACh, and low-resting hearts were restored to the control level with the competitive muscarinic antagonist 10-8 M atropine, whereas excessive exposure of 10-5 M atropine to T4-treated tadpoleheart did not increase heartbeat in spite of the increased frog heartbeat over the control. 10-14 -10-12 M NE increase the tadpole heartbeat in a concentration-dependent manner, however, 10-12 M NE did not act to stimulate adrenergic receptors on both T4-treated tadpole- and the frog-hearts. These results suggest that T4 induces the desensitization of atropine-sensitive muscarinic and adrenergic receptors in organ-cultured tadpole-heart.
Fig. 1. Culture days of st. 57 X. laevis heart in 60% MEM and MHBSS-CM I and II. This experiment was carried out using 4 hearts in 60% MEM and 6 hearts in MHBSS-CM I-II. Values given represent the mean value ± standard error. *Significantly greater (P < 0.05) than the corresponding value for 60% MEM. Long-term culture of spontaneously beating tadpole-hearts. Each medium composition of MHBSS-CM I and II was mentioned in the section of ‘Materials and Methods’.
Fig. 2. Effect of T4 on st. 57 tadpoleheart. Box length indicates the interquartile range or IQR (25th to 75th percentile), and the horizontal line shows the median. The whiskers show distance 1.5 times IQR. This experiment was carried out using 6 hearts. *Significantly greater (P < 0.05) than the corresponding control value.
Fig. 3. ACh-decreased heartbeats frequency of organ-cultured st. 57 hearts. Each value was measured by number of heartbeats times per minute, and represented by ratio to the control value. Experiment was carried out using 4 hearts. Values given represent the mean value ± standard error.
Fig. 4. Effects of ACh and atropine on T4-treated tadpole- and 2–4 weeks frog-hearts. This experiment was carried out using four hearts. Values given represent the mean value ± standard error. Atropine treatments were carried out at 2 min (10−8 M atropine) and 4 min (10−5 M atropine) after ACh treatment. Each value was measured by number of heartbeats times per minute, and represented by ratio given to the control value. Experiment was carried out using 4 hearts. *Significantly less (P < 0.05) than the corresponding value for the rate for the frog heartbeats.
Fig. 5. Effect of NE on in vitro st. 57 hearts in 60% MEM. Each value was measured by number of heartbeats times per minute, and represented by ratio given to the control value. Experiment was carried out using 4 hearts. Values given represent the mean value ± standard error.
Fig. 6. Effect of NE on T4-treated st. 57- and 2–4 weeks frog-hearts after metamorphosis completion. Values given represent the mean value ± standard error. Each value was measured by number of heartbeats times per minute, and represented by ratio given to the control value. Experiment was carried out using 4 hearts. *Significantly less (P < 0.05) than the corresponding values for the ratio given to the value from st. 57 hearts. *Significantly less (P < 0.05) than the corresponding values for the ratio given to the value from st. 57 hearts (control). **Significantly less (P < 0.01) than the corresponding values for the ratio given to the value from st. 57 hearts (control).
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