Relationship between force and Ca2+ concentration in smooth muscle as revealed by measurements on single cells.

Abstract

The role of Ca24 in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2I] simultaneously in individual single smooth-muscle cells. [Ca24] was measured with fura-2 and a high timeresolution dual-wavelength digital microfluorimeter, and force was measured with an ultrasensitive force transducer attached to a probe around which was tied one end of the cell. Both [Ca2+1 and force increase after maximal electrical stimulus, with [Ca24] Calcium is considered the key regulator of contraction of smooth muscle (1, 2), although details of this regulation remain somewhat unclear. The relationship between force and [Ca2 '] has been studied in skinned smooth-muscle strips (3-11) and in smooth muscles that were loaded with aequorin by temporarily permeabilizing the tissue (12). The sensitivity of force to [Ca24] found in chemically permeabilized (skinned) preparations by various investigators varies by almost two orders of magnitude, and several studies showed that the sensitivity can be altered by calmodulin addition (5,6,13) per se, accounts for the several hundred-msec delay between stimulation and onset of active-force production characteristic of smooth muscle. Finally, our results show that the contractile process exhibits decreasing sensitivity to calcium during slow contraction-relaxation cycles. METHODSSmooth muscle cells were isolated from the stomach of the toad Bufo marinus as described (14). Briefly, the procedure involves incubating thin slices of stomach wall first in a dilute solution of collagenase and trypsin and then in a solution of collagenase alone. Cells released from the tissue in this latter solution are collected by mechanically separating the tissue from the cell suspension. Soybean trypsin inhibitor was added to all cell suspensions to suppress any remaining trypsin activity.Cells were loaded with the calcium-sensitive dye fura-2 (15) by incubating them in a solution of the acetoxymethyl ester form ofthe dye, fura-2/AM (Molecular Probes, Eugene, OR). Cells were incubated in this solution at 31°C for 60 min and then returned to room temperature. The amount of fura-2/AM added to the cell suspension was adjusted according to cell density at the ratio of 1.25 x 10-9 mol of fura-2/AM per 106 cells. These loading conditions were found to produce intracellular fura-2 concentrations of 30-100 ,M as estimated by comparing total fluorescence to that from droplets of fura-2 solutions in oil.A dilute suspension of cells that had been loaded with fura-2 was placed on a cover-slip chamber on the stage of an inverted microscope, and the cells were allowed to settle. A single cell was then attached at both ends to two special glass microprobes, each of which had a single anionic-exchangeresin particle cemented to its tip. After attachment of cell to probe, a small section of the muscle cell was wrapped at each end by micromanipulation around the attached probe. The procedure was basically that described by Warshaw and Fay (16). One probe was attached to a micr...

A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells

“…The intracellular acidification could be the consequence of the sustained [Ca'-+]~ rise which leads to H" metabolic generation as previously demonstrated in T cells [38]. Morphological changes shown by electron scanning microscopy of BAEC cultured in the presence of oxidized LDL are consistent with the observed [Ca2"]~ rise which is known to induce cell contraction [39,40] and regulation of endothelial barrier permeability [41]. As oxidized LDL have been detected in atherosclerotic lesions [42], it is not excluded that similar phenomena could occur in vivo, thus raising in the underlying subendothelial space the concentration of LDL and other plasma proteins which are thought to play a role in atherogenesis [43,44].…”

Ca(2+)‐dependent K+ channels of high conductance in smooth muscle cells isolated from rat cerebral arteries.

“…However, [Ca2+]i increases to 6-8 x 10-7M during maximal electrical stimulation of smooth muscle cells (Yagi, Becker & Fay, 1988) and can reach micromolar levels on exposure of these cells to the vasoconstrictor hormone angiotensin II (Capponi, Lew & Valloton, 1986 (Green et al 1991), while the calcium sensitivity of some K+a channels is enhanced in the presence of intracellular modulators, such as Mg2+ (Squire & Petersen, 1987). Thus, a role for the present population of K+a channels in the repolarization of cerebrovascular smooth muscle cannot yet be discounted.…”

Depolarization‐mediated intracellular calcium transients in isolated smooth muscle cells of guinea‐pig urinary bladder.

“…On average, the half-decay time was 0-90 + 0-13 s at -50 and 0-92+0-11 s at -100mV (n=4 Benham, 1989;130 nm in guinea-pig ureter;Benham, 1989b; 140 nm in rabbit ear artery), and with Fura-2 (Yagi et al 1988: 150 nm in toad stomach; Goldman, Wier & Blaustein, 1989: 111 nm in bovine tail artery), or with ion-sensitive electrodes (Yamaguchi, 1986: 160 This peak value is somewhat higher than that reported for similar depolarizations at room temperature in other smooth muscle cells (Aaronson & Benham, 1989: 250 nM;Yagi et al 1988;Becker et al 1989: 400 nM). The difference is most probably due to increase of peak ICa with temperature.…”

“…influx, the rise in [Ca2+]i and the activation of contraction of smooth muscle (Himpens & Somlyo, 1988;Yagi, Becker & Fay, 1988;Becker, Singer, Walsh & Fay, 1989). Applying microspectrofluometry under voltage-clamp conditions allows the pathways for Ca2+ influx to be quantified, e.g.…”