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Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm
Abstract:
Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.
Insights
Rabbit smooth muscle cells contain unidentified anions, as chloride distribution doesn't match osmotic balance. Mitochondria don't regulate calcium in normal cells but accumulate it when damaged.
Area of Science:
- Cellular Physiology
- Biophysics
Background:
- Understanding ion concentrations in smooth muscle is crucial for physiological function.
- Previous studies have not fully elucidated the distribution of ions like potassium, sodium, and calcium within smooth muscle cells and their organelles.
Purpose of the Study:
- To determine the elemental composition of cytoplasm and organelles in rabbit portal-anterior mesenteric vein (PAMV) smooth muscle.
- To investigate the role of mitochondria and nuclei in ion regulation, particularly calcium, in smooth muscle.
Main Methods:
- Electron probe analysis of dry cryosections was employed to measure intracellular ion concentrations.
- Analysis included normal resting muscles, muscles exposed to high KCl, and muscles subjected to K contracture.
Main Results:
- Cytoplasmic concentrations of K, Na, Cl, Mg, Ca, and P were quantified. Intracellular Na + K exceeded cytoplasmic Cl, suggesting non-diffusible anions.
- Mitochondrial calcium content was low in normal fibers and did not increase during K contracture. Damaged fibers showed massive mitochondrial calcium accumulation.
- Nuclear and cytoplasmic Na and Ca levels were similar. Transmitochondrial gradients for K, Na, and Cl were small.
Conclusions:
- The non-Donnan distribution of chloride is not due to organelle sequestration, implying the presence of unidentified non-diffusible anions in smooth muscle.
- Nuclei do not concentrate Na or Ca above cytoplasmic levels.
- Mitochondria do not significantly regulate cytoplasmic Ca in normal PAMV smooth muscle but can accumulate large amounts of Ca in damaged cells.
- In situ transmitochondrial gradients do not support a large electromotive Donnan force for K, Na, and Cl distribution.