Isolation and individual electrical stimulation of single smooth-muscle cells from the urinary bladder of the pig

Abstract

SummaryIn contrast to striated muscle, measurements on strips of smooth muscie cannot be uniquely interpreted in terms of an array of contractile units. Therefore scaling down to the single-cell level is necessary to gain detailed understanding of the contractile process in this type of muscle. The present study describes the development of a method for isolating contractile single smooth muscle cells from pig urinary bladders. Contractile responses evoked by individual electrical stimulation were used as a measure of cell quality during development of the method. Responses were evaluated by measuring latency, contraction and relaxation times, as indicated by visible length changes, and stored on-line in a computer. Initial length, relative shortening and shortening speed were determined by measuring cell lengths in previously timed still video frames using a computer-controlled crosshair device. Increase of stimulus pulse duration resulted in improved responses, indicating that the observed shortening represented a physiological contractile response. Ultimately this method of evaluation was applied to two sets of cell preparations obtained by two different methods, one using only collagenase digestion, the other using mechanical manipulation as well. Both sets showed two main patterns of response to electrical stimulation: a pattern of contraction upon stimulation followed by enhanced contraction when stimulation was switched off (CK), and a pattern of contraction upon stimulation followed by relaxation when the stimulus was switched off (CR).The set of preparations containing the highest percentage of CR cells was found to be superior (i. e. greater initial length, shorter latency and contraction times, increased shortening and higher shortening speed). The method of isolation used for this set gives a high yield of contractile cells available for experimental use over a long span of time.

Mechanical properties of mammalian single smooth muscle cells III. Passive properties of pig detrusor and human a terme uterus cells

“…Conditions of incubation were kept at temperature 37 ~ C; pO 2 150 mm Hg and pCO 2 38 mm Hg approximately, thus resulting in pH 7.35 (Glerum et al, 1987). Cells in suspension were checked for vitality by means of an FDA fluorescence vital staining technique and selected vital cells were knotted between two micropipettes attached to a length displacement apparatus and a micro force transducer (sensitivity 10 #N V 1) as depicted before (Glerum & Van Mastrigt, 1990a, b).…”

“…Both procedures were carried out as described previously (Glerum et al, 1987;Glerum & Van Mastrigt, 1990b). …”

Mechanical properties of mammalian single smooth muscle cells II. Evaluation of a modified technique for attachment of cells to the measurement apparatus

“…Conditions of incubation were kept at temperature 37 ~ C, Po~ 150 mm Hg and Pco2 38 mm Hg approximately, thus resulting in pli 7.35 (Glerum et al, 1987). Fluid evaporation was prevented with a rhin layer of Klearol ( Van Dijk et al, 1984).…”

“…Single smooth muscle cells from a terme human uteruses were obtained likewise from biopsies excised af Caesarean sections. Both procedures were carried out as described previously (Glerum et al, 1987), except for an additional overnight incubation in collagenase at 4 ~ C for both types of tissue and an additional trypsinisation of the pig bladder tissue.…”

“…In the field of muscle contractility research, especially on smooth muscle, there is general agreement on the value and necessity of single cell contractility measurements (Fay et ai., 1976, Glerum et al, 1987. Amongst the essentials needed for such measurements are methods for single cell isolation, a sensitive microforce transducer and a method for cell attachment.…”