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(Circulation Research. 1995;77:220-230.)
© 1995 American Heart Association, Inc.

Articles

Reversible Permeabilization

A Novel Technique for the Intracellular Introduction of Antisense Oligodeoxynucleotides Into Intact Smooth Muscle

Ryan E. Lesh, Andrew P. Somlyo, Gary K. Owens, Avril V. Somlyo

From the Departments of Anesthesiology (R.E.L.), Molecular Physiology and Biological Physics (R.E.L., A.P.S., G.K.O., A.V.S.), Medicine (A.P.S.), and Pathology (A.V.S.), University of Virginia Health Sciences Center, Charlottesville.

Correspondence to Ryan E. Lesh, MD, Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Box 449, Charlottesville, VA 22908.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Antisense oligodeoxynucleotides (ODNs) have been used to modify gene expression in vitro and are also promising therapeutic agents. Although there are numerous reports of antisense ODN–mediated changes in protein expression of cultured cells, use of these compounds to achieve antisense regulation of specific proteins in intact tissue has been limited. The aims of this study were (1) to define organ culture conditions for ileum smooth muscle that would permit long-term maintenance of force-generating capabilities and normal ultrastructure and (2) to develop a method for efficient introduction of antisense ODNs into intact tissue. Sheets of ODN-containing, reversibly permeabilized rat outer longitudinal ileum were maintained in a serum-free organ culture medium for 1 week without significant decreases in tension response to membrane depolarization or carbachol stimulation; the G protein–coupled calcium sensitization pathway was also intact after 7 days. Reversible permeabilization, a method previously used to load smooth and cardiac muscle with aequorin and heparin, was effective for loading >95% of ileum smooth muscle cells with a fluorescein-conjugated antisense ODN (5'-AAGGGCCATTTTGTT-FITC-3'). Confocal microscopy of reversibly permeabilized smooth muscle loaded with fluorescent antisense ODNs revealed intense nuclear fluorescence and less intense, homogeneous, cytoplasmic fluorescence. Internally radiolabeled ODNs (homologous to the above sequence) showed complete degradation between 4 and 16 hours after introduction into the cells. In summary, we have demonstrated methods for long-term organ culture and high-efficiency introduction of antisense ODNs into intact smooth muscle sheets. Such methods have broad potential utility for investigating many questions in smooth muscle biology. At present, however, a major limitation of this approach is the short half-life of phosphorothioated ODNs.

Key Words: antisense oligodeoxynucleotides • smooth muscle • reversible permeabilization • confocal microscopy • electron microscopy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The value, as well as the limitations, of ODNs as modifiers of gene expression and therapeutic agents is increasingly being recognized, and ODNs complementary to specific nucleotide sequences of pre-mRNA or processed mRNA ("antisense" ODNs) are being introduced into cells to target specific genetic sequences for selective inhibition of protein expression (reviewed in References 1 through 9^{1 2 3 4 5 6 7 8 9} ). The utility of antisense approaches for studying the role of specific intracellular proteins has been demonstrated in cultured cells, and there are numerous reports of antisense ODN–mediated effects on cellular protein expression by antisense ODNs added to the cell culture medium. Recent work has shown that cellular uptake of ODNs can be enhanced by linkage to cholesterol moieties or to DNA-binding molecules such as acridine^{10 11} or by delivery of ODNs with phospholipid vesicles¹² or cationic lipids.^{13 14 15} There is limited information, however, about the use of antisense ODNs to study physiological processes in intact tissues, apart from investigations of injected intraperitoneal antisense ODNs, which affect primarily the liver,¹⁶ and catheter introduction of antisense ODNs into localized regions of vascular smooth muscle after balloon angioplasty.¹⁷

The development of methods suitable for efficient intracellular targeting of ODNs in intact tissues is particularly important for therapeutic uses and for determining their effects on functions, such as contractility, that are not normally expressed and/or readily measured in cultured cells. The potential therapeutic value of ODNs for the inhibition of myointimal thickening due to abnormal migration and proliferation of vascular smooth muscle cells has already been demonstrated experimentally,^{17 18} and optimization of intracellular targeting may lead to successful applications in humans. In the case of smooth muscle, the effect on contractile properties of knocking out putative regulatory proteins would be extremely valuable for directly testing suggested regulatory mechanisms. This goal, however, is not readily achieved with cells that dedifferentiate in culture to noncontracting phenotypes.

The aim of the present study was to define conditions of organ culture for maintaining normal contractile function of our smooth muscle preparations and to develop a method for the efficient introduction of antisense ODNs into intact smooth muscle bundles. In this initial study, we used ileum smooth muscle, because its consistent, spontaneous contractile activity and responses to both depolarization and agonists are useful indexes of normal function. We show that ileum can be maintained in organ culture for up to 7 days with undiminished contractile responses and normal ultrastructure; in addition, reversible permeabilization of intact sheets of ileum permits highly efficient nuclear loading of cells with fluorescent antisense ODNs. Our experiments with internally radiolabeled, phosphorothioated antisense ODNs showed relatively rapid degradation of the ODNs. This finding underscores the importance of directly assessing the intracellular lifetime of antisense ODNs and emphasizes the need for more stable antisense constructs for this approach to be useful for studying the biological function of proteins with half-lives greater than several hours.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
ODN Preparation
ODNs were prepared commercially by the Biomolecular Research Facility at the University of Virginia, Integrated DNA Technologies, Inc, or Oligos, Etc, Inc. ODNs supplied by the Biomolecular Research Facility contained 3,5' and 3,3' phosphorothioate modifications to enhance their tissue half-life and were prepared on a Biosearch model 8700 DNA synthesizer (Biosearch, Inc) by use of standard cyanoethyl phosphoramidite chemistry. All ODNs were purified with Poly-Pak cartridges (Glen Research), and the resulting purity was checked by polyacrylamide gel capillary electrophoresis. The ODNs were supplied in a lyophilized form, reconstituted in sterile water as a 10 mmol/L stock solution, and stored at -20°C until use. ODNs obtained from Integrated DNA Technologies contained 3,5' and 3,3' internucleoside methoxyethylphosphoramidate linkages and were purified on a Sephadex G25 column (Pharmacia), and their purity was assessed by electrophoresis on an 18% polyacrylamide gel. ODNs supplied by Oligos, Etc, Inc also contained 3,5' and 3,3' internucleoside phosphorothioate modifications. They were purified by reversed-phase high-performance liquid chromatography, and their purity was assessed by polyacrylamide gel electrophoresis. Phosphoramidate ODNs were supplied in a lyophilized form, reconstituted with sterile water as a 1 mmol/L stock solution, and stored at -20°C. In addition to antisense ODNs, one sense and one scrambled construct were synthesized for each antisense sequence; the scrambled sequence contained, in random order, the same base composition and internucleoside modifications as the antisense sequence. A 5' fluorescein-conjugated antisense ODN, which contained identical 3' and 5' internucleoside modifications, was synthesized for fluorescence microscopy. Fig 1 shows the sequences and modifications of ODNs used in these experiments. The antisense construct was designed to the translation-initiation codon of the rat type 1 Ang II receptor.¹⁹

View larger version (20K): [in this window] [in a new window]   Figure 1. ODN sequences. ODNs were synthesized in sense and antisense orientations, and as a scrambled sequence, to the published type 1 (vascular) Ang II receptor cDNA sequence. Asterisks in the sequence indicate the positions of either the phosphorothioate or methoxyethylphosphoramidate internucleoside modifications. Fluorescein (Fl) was conjugated to the 5' end of the fluorescent antisense ODN.

Radioactive Labeling of ODNs
A phosphorothioate-modified antisense–Ang II ODN was internally radiolabeled with ³²P (as described in References 20 and 21^{20 21} ) to estimate the duration of its intracellular stability. An 8-base ODN (5'-AT TTT *G*T*T-3', where * designates an internucleoside phosphorothioate modification) was 5' end-labeled with -³²P-ATP (6000 Ci/mmol, New England Nuclear) and T4 polynucleotide kinase (Boehringer Mannheim). The 5' end-labeled 8-base ODN was then ligated overnight to a 7-base ODN (5'-A*A*G*GGCC-3') at room temperature with T4 DNA ligase (Boehringer Mannheim); this reaction contained a complementary 21-base DNA template (5-TAT AAC AAA ATG GCC CTT GCG-3'). The ligation product (³²P-labeled 15-base ODN) was isolated from the template and unligated ODNs by polyacrylamide gel electrophoresis on a 17.5% polyacrylamide/7 mol/L urea gel, eluted from the gel matrix with 0.1 mol/L ammonium bicarbonate, and purified on a Nensorb 20 cartridge (Dupont/New England Nuclear) according to the manufacturer's recommendations. The radiolabeled ODN was eluted from the Nensorb column with 50% ethanol, dried by vacuum evaporation in a SpeedVac Concentrator (Savant), and reconstituted with sterile, deionized water. Specific activity of the labeled ODN was 0.8 µCi/mmol.

Intracellular Stability of ODNs
The intracellular stability of the ODN used in these experiments was estimated by introducing the internally labeled phosphorothioate-modified antisense–Ang II ODN into ileum strips by the reversible permeabilization protocol described below and mounting it for tension measurements in an organ bath containing SFM gassed with 95% O₂/5% CO₂ at 37°C. Tissue samples were removed from the bath, frozen in liquid nitrogen at 1, 2, 4, and 16 hours, and stored for 24 hours at -80°C. These strips were homogenized on ice in 0.1 mol/L Tris buffer, pH 7.3, containing 1% SDS, then extracted with an equal volume of phenol and centrifuged in a microcentrifuge, and the aqueous layer was decanted (as described in Reference 22²² ). The aqueous layer was mixed with an equal volume of gel loading buffer to contain a final concentration of 50% formamide, 0.5x Tris-borate-EDTA, and 0.05% bromophenol blue; this mixture was heated to 85°C for 2 minutes and rapidly cooled on ice before loading onto a 17.5% polyacrylamide/7 mol/L urea gel for electrophoresis in Tris-borate-EDTA buffer. After electrophoresis, the gel was fixed in 10% methanol/10% acetic acid, dried, and exposed on x-ray film (Fuji RX medical x-ray film).

Fluorescent Dextrans, Albumin, ß-Galactosidase, and Immunoglobulins
Rhodamine B–conjugated dextrans of 40 kD and 70 kD were obtained from Molecular Probes. Both fluorescent dextrans were reconstituted with water to a 100-mg/mL stock solution and added to reversible permeabilization solutions 2 through 4 (see below) to a final concentration of 500 µg/mL. Stock solutions of fluorescent dextrans were kept in the dark at 4°C.

FITC-conjugated albumin and FITC-conjugated ß-galactosidase were purchased from Sigma Chemical Co. Both proteins were reconstituted to a 1 mmol/L stock solution with water and stored at -20°C until use. The final concentration of fluorescent albumin and ß-galactosidase was 5 µmol/L in reversible permeabilization solutions 2 through 4. To exclude contamination of these proteins by free FITC, stock solutions of both the FITC-labeled albumin and FITC-labeled ß-galactosidase were washed in 10-kD molecular weight cutoff ultrafree-MC centrifugal filtration units (Sigma Chemical Co).

TRITC-conjugated immunoglobulins [goat anti-rat IgG and goat anti-rat F(ab')₂] were obtained from Jackson Immuno-Research. The lyophilized proteins were reconstituted with sterile water to a 10 mmol/L stock solution, which was diluted to a final concentration of 100 µmol/L in reversible permeabilization solutions 2 through 4.

Tissue Preparation
Rectangular pieces of the longitudinal layer of ileum smooth muscle were obtained from adult male rats and adult male guinea pigs (purchased from Hilltop Laboratory Animals). Guinea pig ileum was used in initial experiments; however, rat ileum was used later because of the greater availability of rat cDNA sequences for designing species-specific antisense ODNs. Euthanasia and animal-handling procedures were in accordance with institutional policies and conformed to Public Health Service guidelines regarding the humane use and care of laboratory animals. Before tissue harvest, the animals were anticoagulated with heparin 500 U IP administered approximately 20 minutes before they were killed, then exsanguinated after a halothane overdose. Once the peritoneal cavity was opened, the ileum was kept moist with a solution of HEPES-buffered modified Krebs' solution warmed to 37°C. The lumen of the ileum was flushed with warm buffer, and 1.2- to 1.5-cm segments were cut with dissecting scissors. The outer longitudinal and several layers of the inner circular smooth muscle were removed from the ileum segments, as previously described.²³ Briefly, segments of ileum were gently pulled onto a 6-mm-diameter glass pipette, and the outer muscle layers were scored longitudinally with a razor blade on both sides of the remaining mesentery. Small sheets of outer longitudinal muscle were gently teased free from the underlying circular muscle and mucosa. These muscle sheets (measuring 7 mm wide and 15 mm long) were pinned onto small strips of silicone rubber (Sylguard, Dow Corning Corp) with 0.1-mm stainless steel minuten pins and allowed to equilibrate in HEPES-buffered Krebs' solution at 37°C for 30 to 60 minutes. All muscle sheets used in these experiments showed return of normal spontaneous contractile activity.

Reversible Permeabilization
ODNs were introduced into smooth muscle cells reversibly permeabilized by a method that was previously used to introduce fluorescent heparin,²⁴ a modification of the method originally described for the intracellular loading of aequorin.^{25 26 27 28} Sheets of ileum were incubated in HEPES-buffered Krebs' solution at 37°C before reversible permeabilization, according to the protocol; incubation times, temperatures, and solution constituents (in mmol/L) were, for solution 1, 20 minutes in EGTA 10, KCl 120, ATP 5, MgCl₂ 2, TES 20 (pH 6.8 at 2°C); for solution 2, 90 minutes in 10 µmol/L ODN, KCl 120, ATP 5, MgCl₂ 2, TES 20 (pH 6.8 at 2°C); for solution 3, 30 minutes in 10 µmol/L ODN, KCl 120, ATP 5, MgCl₂ 10, TES 20 (pH 6.8 at 2°C); and for solution 4, 30 minutes in 10 µmol/L ODN, NaCl 140, KCl 5, MgCl₂ 10, glucose 5.6, MOPS 2 (pH 7.1 at 22°C).

Strips to be loaded with ODNs were exposed to a final concentration of 10 µmol/L ODN in solutions 2, 3, and 4. After the 30-minute incubation in solution 4, CaCl₂ was added at 15-minute intervals over a period of 1 hour to give the following final calcium concentrations, in mmol/L: 0.001, 0.01, 0.1, and 1.6. After the external Ca²⁺ concentration was raised, strips were transferred to HEPES-buffered Krebs' solution at 37°C for 30 minutes. For control experiments, the muscle strips were exposed to the reversible permeabilization protocol without addition of ODN or fluorescent probe to any of the solutions.

In one experiment, three different concentrations of a fluorescein-labeled 11-mer (100 nmol/L, 1 µmol/L, and 10 µmol/L) were introduced into reversible permeabilization solutions 2 through 4 to determine whether the distribution of fluorescent ODNs was concentration dependent. This ODN was synthesized by Operon Technologies, Inc.

Permeabilization of Ileum Strips With Staphylococcal -Toxin
Small strips (1.5 mm wide, 4 to 5 mm long) dissected from a larger sheet of reversibly permeabilized ileum that had been in organ culture for 7 days were used to determine whether intracellular signaling processes were retained. The strips were mounted on isometric tension transducers (AE801, Akers) in chambers equipped with a bubble plate, as previously described.²⁹ After baseline tension measurements had been obtained by stimulation of the strips with high [K⁺], carbachol, and Ang II, strips were permeabilized by incubation at 22°C with 1 to 2 mg/mL of 15 to 20 µg/mL Staphylococcus aureus -toxin (List Biological Laboratories) in pCa 6.5 intracellular solution.³⁰ The permeabilized strips were washed in solution with pCa >8, and the calcium ionophore A23187 was applied at a final concentration of 10 µmol/L to deplete internal calcium stores. To demonstrate that receptor-mediated, G protein–coupled calcium sensitization was retained in organ-cultured reversibly permeabilized muscles, the permeabilized strips were partially contracted with buffered calcium (pCa 6.7) and, at the tension plateau, 5 µmol/L GTP followed by 100 µmol/L of carbachol was added. The supramaximal concentration of carbachol was used to reduce diffusional delays. Subsequently, a supramaximal concentration of GTP--S was added to estimate the maximum G protein–mediated calcium sensitization response. The strip was finally exposed to pCa 4.5 solution to elicit the maximum contractile response to calcium. Calcium was washed out with 1 mmol/L EGTA relaxing solution to return the muscles to baseline tension.

Confocal Microscopy
A Zeiss Axiovert 35-BioRad MRC-600 or MRC-1000 laser scanning confocal imaging system equipped with a krypton-argon laser and an Olympus x40, numerical aperture=1.3 lens was used to determine the efficiency of loading intracellular fluorescent ODNs, dextrans, albumin, ß-galactosidase, and immunoglobulins. Confocal images were obtained throughout the thickness of the tissue. The laser was fitted with a blue filter block (excitation maximum, 488 nm) to image the fluorescein-conjugated compounds, and a yellow filter block (excitation maximum, 568 nm) was used to image smooth muscle cells loaded with rhodamine-conjugated compounds.

Organ Culture
After reversible permeabilization and introduction of ODNs, the sheets of longitudinal ileum smooth muscle were mounted in 45-mL glass muscle chambers. All solutions bathing the muscle were gassed with a stream of 95% air/5% CO₂ and maintained at 37°C by continuous water circulation through the outer jacket of the chamber. Muscle sheets were mounted in the chamber by tying two corners of the muscle to a stationary bar with 5-0 surgical silk; the remaining two corners were also tied to a triangular stainless steel mount with surgical silk. The apex of the triangle was then attached with 5-0 surgical silk to an isometric force transducer (Grass FT03, Grass Instrument Co) interfaced with a Gould universal amplifier (Gould Inc), and tension traces were displayed on a Gould TA 2000, eight-channel chart recorder. Alternatively, the two long ends of the muscle sheet were clamped between loops of a precut 4.5-mm-diameter stainless steel spring; one end was looped through the stationary bar, and the other was tied with surgical silk to the tension transducer. The tissue was gently stretched to 1.2 times its resting length. The muscle strips were maintained by incubation in SFM at 37°C gassed with 95% air/5% CO₂; the SFM was changed twice daily, after each agonist-stimulation protocol. The composition of the SFM used in these experiments was as follows: Dulbecco's minimal essential medium+F12 at a 1:1 ratio, penicillin/streptomycin 1%, L-glutamine 200 mg/L, L-ascorbic acid 35.2 mg/L, transferrin 5 mg/L, selenium 3.25 µg/L, and insulin 2.85 mg/L. At 37°C, gassed with filtered (Millipak 60, Millipore Corp) 95% air/5% CO₂, the SFM had a pH of 7.4.

Tension responses of organ-cultured strips to high [K⁺] (140 mmol/L), carbachol (10 µmol/L), and in some experiments, Ang II (10 nmol/L) were measured twice daily. The muscle chamber was drained of SFM and washed with 35 mL of HEPES-buffered Krebs' solution; this buffer was then replaced with prewarmed normal Krebs' solution. Once the muscle tension had returned to baseline, the chamber was again drained and filled with 25 mL of warm HEPES-buffered Krebs' solution containing 10 µmol/L Ang II. The Ang II was allowed to remain in contact with the strip for 3 to 5 minutes, and the muscle chamber was washed with 2 volumes of warmed buffer. When the tension returned to baseline, carbachol was introduced from a 10 mmol/L stock solution into the muscle chamber at a final concentration of 10 µmol/L. Tension was recorded for 5 minutes, and the strip was washed twice with warm buffer. At the end of this stimulation protocol, the HEPES-buffered Krebs' solution was replaced with fresh SFM, and the strips were maintained in this solution until the next stimulation. Stock solutions of carbachol and Ang II were made in deionized, distilled water and kept frozen at -20°C.

Electron Microscopy
Samples of rat ileum were reversibly permeabilized and maintained in organ culture for up to 7 days. Strips were removed from culture at time 0 and 1, 3, 5, and 7 days after reversible permeabilization and were fixed in 2% glutaraldehyde, either overnight at 4°C or for 1.5 hours at room temperature. They were postfixed with 2% OsO₄ in sodium cacodylate buffer and then 1% tannic acid at pH 7.4. Subsequently, they were stained en bloc with saturated aqueous uranyl acetate and dehydrated in a series of increasing ethanol concentrations, then embedded in Spurr's resin. Sections were cut with a diamond knife to 90-nm thickness and stained with lead citrate. Micrographs were obtained on a Philips CM-12 electron microscope.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organ Culture of Reversibly Permeabilized Ileum
The initial aims of this study were (1) to define the conditions of organ culture that permit long-term maintenance of normal contractile function and muscle ultrastructure and (2) to develop a method for high-efficiency intracellular delivery of ODNs into intact tissue. Sheets of reversibly permeabilized outer longitudinal ileum smooth muscle were maintained in organ culture for up to 7 days, and tension responses to agonist stimulation (carbachol) and membrane depolarization (high [K⁺]) were sampled twice daily. Small samples of muscle were fixed at 2-day intervals to determine the effects of organ culture on the muscle ultrastructure.

The rat Ang II receptor was selected as the target for knockout in these experiments because the cDNA sequence was available for this receptor and selective receptor agonists and antagonists also exist. Preliminary experiments demonstrated that the contractile response to Ang II in ileum strips was completely abolished by the selective type 1 Ang II receptor antagonist Dup 753 (Du Pont Merck Pharmaceutical Co) (data not shown), which suggested that the tension response to this agonist was mediated through the type 1 Ang II receptor.

Tension responses were measured in two groups of muscle: control preparations that were reversibly permeabilized without the addition of ODNs and experimental preparations that were reversibly permeabilized with the introduction of either scrambled or antisense ODNs. In both groups, normal spontaneous contractions appeared 30 minutes after transfer from calcium-containing solution 4 to normal HEPES-Krebs' solution at 37°C. Each muscle sheet showed vigorous contractile responses to depolarization with high [K⁺] and to stimulation with carbachol. Fig 2 demonstrates the typical time course of tension normalized to the initial responses to K⁺ and carbachol; the amplitude of force is plotted versus hours after reversible permeabilization. The amplitude of carbachol-induced contractions was consistently higher than the K⁺-induced contracture both before and after reversible permeabilization and in organ culture. Representative K⁺-induced contractures and responses to carbachol are shown in Fig 2 and indicate no significant decrease over 72 hours. The tension generated by reversibly permeabilized muscle strips was of a magnitude similar to that developed by nonpermeabilized ileum muscle strips.²⁴ The ileum also retained its phasic response to membrane depolarization; tension responses to carbachol were more tonic, as also found in non–organ-cultured ileum.²³ Tension responses to Ang II diminished over the first 24 hours of organ culture in both antisense-treated and control strips (n=3 experiments). Similar decreases in tension response to Ang II stimulation in both control (reversibly permeabilized without addition of ODN) and antisense ODN preparations suggest that there was a spontaneous reduction in Ang II responsiveness in organ culture.

View larger version (19K): [in this window] [in a new window]   Figure 2. Graphs showing normalized peak tensions developed for four sheets of reversibly permeabilized (RP) ileum measured during 75 hours in organ culture. A, Plot of the peak tensions (mean±SD) normalized to the initial (t=0 hours) peak potassium-induced tension. The mean of normalized peak tensions is calculated from three sheets of ileum that contained either an antisense (n=2) or scrambled (n=1) ODN. The control is a reversibly permeabilized ileum sheet into which no ODN was introduced. B, Plot of normalized peak carbachol-induced tensions in the same four muscle sheets. Data are also plotted as the mean of the peak tensions (±SD) normalized to the initial carbachol-induced contraction.

Force records obtained from a reversibly permeabilized strip of muscle maintained for 7 days in organ culture are illustrated in Figs 3 through 6 and show the contractile responses to high [K⁺] (140 mmol/L K⁺), carbachol (10 µmol/L), Ang II (doses of 10 nmol/L, 100 nmol/L, and 1 µmol/L), and endothelin (10 nmol/L and 1 µmol/L). Carbachol-induced tension responses in the ODN-containing strips and the reversibly permeabilized strips without intracellular ODNs were not significantly different. Responses to endothelin and Ang II were seen only at concentrations of 1 µmol/L for endothelin (reported ED₅₀ in guinea pig ileum is 1 nmol/L³¹ ) and at 100 nmol/L for Ang II, in contrast to tension responses at lower concentrations in noncultured strips, suggesting a decrease in sensitivity to these two agonists but not to carbachol.

View larger version (22K): [in this window] [in a new window]   Figure 3. Tension traces obtained at 37°C from a small, intact strip of ileum 7 days after reversible permeabilization. The phasic response to potassium-induced membrane depolarization was, characteristically, of smaller amplitude than the peak response to carbachol. No response is demonstrated to 10 nmol/L endothelin; however, 1 µmol/L endothelin added to HEPES-buffered normal Krebs' solution stimulated clusters of spontaneous contraction similar in peak tension to the potassium contraction. This activity remained after the strip was washed in warm buffer.

View larger version (56K): [in this window] [in a new window]   Figure 4. Tracing showing spontaneous contractions of a strip of intact ileum in HEPES-buffered Krebs' solution at 37°C 7 days after reversible permeabilization and maintenance in organ culture.

View larger version (17K): [in this window] [in a new window]   Figure 5. Tracing showing contractions of intact ileum smooth muscle induced by 100 nmol/L Ang II 7 days after reversible permeabilization and organ culture.

View larger version (12K): [in this window] [in a new window]   Figure 6. Tension responses measured at 22°C in an -toxin–permeabilized ileum strip 7 days after reversible permeabilization. Calcium stores were depleted with A23187. A characteristic phasic response to pCa 6.7 is noted; carbachol (a known calcium-sensitizing agent) induces a rise in tension with [Ca2+] clamped at pCa 6.7. Supramaximal stimulation of the G protein–coupled calcium sensitization pathway is seen on addition of 300 µmol/L GTP--S, and maximal calcium-induced tension is elicited with pCa 4.5 solution.

To assess the effect of organ culture on the calcium sensitivity of the contractile apparatus, the same muscle strips were permeabilized with -toxin and stimulated with buffered calcium at pCa 6.7. Fig 6 demonstrates the responses to calcium at pCa 4.5 and pCa 6.7 and the increase in calcium sensitivity induced by carbachol. GTP--S (300 µmol/L) stimulated maximal G protein–mediated calcium sensitization. These data demonstrated that receptor-coupled responses to carbachol, Ang II, and endothelin were present and that the G protein–coupled calcium sensitization pathway (reviewed in Reference 32³² ) was also intact after 7 days in organ culture. In Fig 4, spontaneous contractions are seen after the washout of carbachol and incubation in HEPES-buffered Krebs' solution.

The effect of long-term culture and of the reversible permeabilization procedure on muscle ultrastructure showed that in three of the four sets of tissue, the ultrastructure was essentially normal throughout the 7-day period of organ culture (Figs 7 and 8). The muscle cells had a normal spindle shape, the surface membrane contained caveolae, and the sarcoplasmic reticulum showed no evidence of swelling. Nuclei, mitochondria, and thin and thick filaments were normal and were indistinguishable from untreated tissue apart from the presence of occasional lysosomes on day 3 in organ-cultured cells (Fig 7).

View larger version (167K): [in this window] [in a new window]   Figure 7. Electron micrograph of ileum maintained in organ culture for 3 days after reversible permeabilization. At 3 days, there appears to be an increase in the number of lysosomes (L) present in the cells; otherwise, the ultrastructure is normal. M indicates a mitochondrion.

View larger version (164K): [in this window] [in a new window]   Figure 8. Electron micrograph of ileum maintained in organ culture for 7 days after reversible permeabilization. At 7 days in organ culture, both outer longitudinal and inner circular smooth muscle retain their normal arrays of thin and thick (arrows) filaments; there may be a slight increase in the intermediate filaments in several images.

In one experiment, the cytoplasm of some cells contained electron-dense accumulations consistent with myosin aggregates; normal arrays of myosin and actin filaments were not present. These "tactoids" appeared in many of the smooth muscle cells of this one strip between days 3 and 5 in culture. These cells also contained a notably abundant rough endoplasmic reticulum.

Intracellular Localization of Fluorescent ODNs
To introduce ODNs into smooth muscle, sheets of outer longitudinal ileum smooth muscle were reversibly permeabilized and exposed to fluorescein-conjugated phosphorothioate ODNs while permeable (see "Methods"). The intracellular localization and efficacy of ODN incorporation was determined by confocal microscopy. As seen in Figs 9 through 11, the nuclei of both the longitudinal and circular smooth muscle layers were brightly fluorescent; the nuclear signal was much more intense than the nearly homogeneous, cytoplasmic fluorescence; in higher-magnification views (Fig 10), the nuclear fluorescence was inhomogeneous. Fluorescent ODNs were present in a very high percentage (>95%) of cells (see Figs 9 through 11), and the frequency and distribution of nuclei were comparable to strips treated with ethidium bromide (to stain all nuclei).

View larger version (73K): [in this window] [in a new window]   Figure 9. Confocal photomicrographs of reversibly permeabilized longitudinal smooth muscle layer of the ileum showing the presence of fluorescent signal in the nuclei and cytoplasm. An unmodified, fluorescent, antisense ODN was introduced into these cells with a high degree of efficiency. As a control (B), 10 mmol/L free fluorescein was loaded into reversibly permeabilized smooth muscle; minimal labeling is seen in an image collected with the same parameters. The fluorescent streak in the lower right-hand corner is thought to represent a nerve of the autonomic plexus present in the muscle layers of the ileum.

View larger version (45K): [in this window] [in a new window]   Figure 10. High-magnification confocal images of reversibly permeabilized (A) outer longitudinal and (B) inner circular smooth-muscle layers of the ileum. A fluorescent phosphorothioate-modified antisense ODN was introduced in A and an unmodified fluorescent antisense ODN in B. Labeling is predominantly localized to nuclei, where it appears inhomogeneous.

View larger version (91K): [in this window] [in a new window]   Figure 11. Confocal micrographs of reversibly permeabilized ileum at four time points after reversible permeabilization and introduction of a fluorescent ODN: 0, 24, 48, and 72 hours. A, Image (obtained at 0 hours) of the outer longitudinal layer of the ileum with the characteristic appearance of abundant nuclear fluorescence and a high efficiency of cell labeling. B, Similar image taken approximately 24 hours after reversible permeabilization and maintenance in organ culture. A pattern of labeling similar to that in A is seen. At 48 hours in culture (C), there is a somewhat patchy accumulation of fluorescent signal; in the cytoplasm, however, the nuclei still contain discernible fluorescent material. D, Images at 72 hours show streaky cytoplasmic fluorescence and inhomogeneous nuclear labeling. The number of cells that contain fluorescent signal appears somewhat less than at earlier times. Confocal images sampled throughout the depth of the preparations were identical, illustrating the high efficiency of tissue labeling.

During the initial 3 days, nuclear fluorescence was greater than cytoplasmic, although somewhat fewer cells contained fluorescent nuclei on day 3 than immediately after reversible permeabilization. Fig 11A through 11D are images obtained at 0, 24, 48, and 72 hours after reversible permeabilization. The characteristic pattern of nuclear localization is seen at 0 hours, with much more intense nuclear than cytoplasmic fluorescence. At 24 hours, there appears to be no significant decrease in either the nuclear or cytoplasmic fluorescence, and the number of cells containing fluorescent signal was similar to that at 0 hours. In three experiments, at 24 hours the morphology of the cells and the fluorescent labeling were indistinguishable from the original images. By 48 hours, cytoplasmic fluorescence increased and became more punctate and inhomogeneous, although nuclear fluorescence was still more intense than cytoplasmic. By 72 hours, there was a change in the appearance of the nuclear and cytoplasmic fluorescence: nuclear fluorescence appeared somewhat lobulated in higher-magnification views, and the pattern of cytoplasmic fluorescence was inhomogeneous with, in some places, a lacy appearance.

One experiment was performed to determine the concentration dependence of intracellular ODN distribution and loading efficiency. Three different concentrations of FITC-labeled ODN (100 nmol/L, 1 µmol/L, and 10 µmol/L) were added during the reversible permeabilization protocol. Ileum reversibly permeabilized in the presence of each ODN concentration showed similar patterns of distribution and the same efficiency of cellular uptake: nuclear fluorescence predominated, and >95% of the cells were loaded. Estimates of ODN loading were made by confocal microscopy in areas of 87 000 µm² using identical image collection parameters. Mean pixel intensities and measures of total fluorescence varied linearly with the ODN concentration in the reversible permeabilization solution over the range of concentrations tested.

Experiments designed to estimate the loading efficiency and molecular weight exclusion during reversible permeabilization showed that reversibly permeabilized guinea pig ileum strips were permeable to both 40-kD and 70-kD rhodamine B–conjugated dextrans. The distribution of fluorescence in the cytoplasm of reversibly permeabilized ileum exposed to either 40-kD or 70-kD fluorescent dextran was relatively homogeneous, and both the cytoplasm and nucleus were fluorescent (Fig 12). In control experiments, in which strips of smooth muscle were incubated without being reversibly permeabilized in temperature-matched HEPES-buffered Krebs' solution containing either the 40-kD or the 70-kD dextran probe, only extracellular fluorescence was detected by confocal microscopy. Reversibly permeabilized smooth muscle cells loaded with fluorescent dextrans retained the fluorescent cytoplasmic signal for 24 hours or longer in organ culture; however, during that time, vacuoles that excluded fluorescent signal appeared in some of the cells (Fig 12). There appeared to be no preferential nuclear or subcellular accumulation of fluorescence during the time these strips were maintained in culture. Fluorescein-conjugated albumin was also incorporated into both the cytoplasm and the nuclei of rat ileum smooth muscle cells during reversible permeabilization. Representative images of albumin-loaded cells are seen in Fig 13A. In cells loaded with FITC-albumin, nuclear fluorescence was greater than cytoplasmic, compared with either of the fluorescent dextrans (see Fig 13B): albumin appeared to concentrate in the nucleus, whereas dextran was uniformly distributed throughout the cytoplasm and nuclei.

View larger version (115K): [in this window] [in a new window]   Figure 12. Confocal micrograph of guinea pig ileum 24 hours after reversible permeabilization and loading with a 40-kD fluorescent dextran. Many cells retain the fluorescent signal, although multiple cytoplasmic vacuoles (V) appear in several of the cells. The nuclei are not selectively labeled with dextran, and homogeneous fluorescence is present throughout the cytoplasm.

View larger version (42K): [in this window] [in a new window]   Figure 13. A, Reversibly permeabilized rat ileum with cytoplasmic and nuclear uptake of FITC-albumin. The nuclei contain greater fluorescent signal compared with the cytosol, and the majority of cells are labeled. B, Confocal micrograph of the outer longitudinal layer of rat ileum after reversible permeabilization in the presence of a rhodamine B–conjugated goat anti-rat IgG. The extracellular boundaries of the cell are seen; however, there is no detectable intracellular fluorescent signal. The streaks of fluorescence seen on the lower left of the image reflect en face views of the extracellular space.

Recovery of Radiolabeled Oligonucleotides
To determine the intracellular lifetime of the antisense ODN used in the above experiments, we internally radiolabeled the ODN, introduced the radiolabeled ODN into ileum by reversible permeabilization, and recovered the ODN from the tissue samples at different times. Autoradiograms of radiolabeled ODNs recovered from ileum smooth muscle cells at 1, 2, 4, and 16 hours after reversible permeabilization are shown in Fig 14. Bands that comigrate with the control 15-mer are visible at 1, 2, and 4 hours in organ culture. By 16 hours after reversible permeabilization, however, no full-length ODNs (or degradation products) are visible with a 48-hour autoradiographic exposure. In other experiments (data not shown), in which whole-cell homogenates were electrophoresed without prior phenol extraction, radioactive signal was present at the gel origin at times >2 hours after reversible permeabilization, similar to the radiolabeled ODNs shown by others to remain in the gel loading wells.³³ Our results show that no detectable, full-length 15-mer remains by 16 hours after introduction of ODNs into cells by reversible permeabilization.

View larger version (81K): [in this window] [in a new window]   Figure 14. Autoradiogram of radiolabeled ODNs recovered from rat ileum strips 1, 2, 4, and 16 hours after their introduction by reversible permeabilization. The control lane, labeled C, is radioactive ODN recovered from tissues incubated in HEPES-buffered Krebs' solution containing ODNs; temperatures and incubation times matched those for tissues incubated in the reversible permeabilization solutions. The lane labeled 0l is internally radiolabeled 15-mer, which marks the migration distance of the starting 15-mer in this gel matrix. Note that there are no degradation products of recovered ODNs at 72 hours of exposure on this autoradiogram, and no signal appears at the 16-hour time point.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of this study show the feasibility of efficient introduction of ODNs into cells in intact tissue, the predominantly nuclear localization of these ODNs, and the maintenance of normal ultrastructure and contractility of smooth muscle strips containing ODNs for at least 7 days in organ culture.

An FITC-labeled antisense ODN complementary to the translation initiation codon (AUG) of the type 1 Ang II receptor mRNA was introduced by reversible permeabilization into >95% of the ileum smooth muscle cells. Laser-scanning confocal microscopy of FITC-labeled antisense ODNs showed the highest concentration in the nuclei, a result consistent with other reports demonstrating preferential nuclear accumulation of antisense ODNs after microinjection.^{34 35 36 37} Incubation of cultured cells in ODN-containing culture medium may also result in nuclear targeting^{38 39} but more frequently results in endosomal localization. Specific nuclear proteins that bind ODNs in vitro may provide one of the non-RNA, non-DNA nuclear binding sites³⁴ ; nonspecific protein binding of polyanionic phosphorothioate ODNs has also been observed.⁷ Nuclear histones may play a role in nuclear localization, since they are a binding site for fluorescent heparin, also a polyanion.^{24 40} The endosomal localization of cytoplasmic antisense ODNs, mediated by adsorptive endocytosis of ODNs by nonpermeabilized cells,^{41 42 43 44} is circumvented by reversible permeabilization (present study). Bulk adsorptive endocytosis of ODNs also requires energy and is consequently temperature sensitive, whereas in our experiments tissues were incubated in ODN-containing reversible permeabilization solutions at 4°C, a temperature too low to support endocytosis. Differential localization of internalized ODNs to either the cytosol or nucleus may be cell-type specific.¹¹ The targeting of ODNs to either the nucleus or the cytoplasm, however, does not identify the site of antisense effect, since there are reports implicating both nuclear and cytoplasmic mechanisms in the arrest of protein synthesis.⁴ In the present study, fluorescence was detected in the cytoplasm, albeit at a much lower intensity than the nucleus, consistent with either or both as sites of action of ODNs.

The ultrastructural features and excitation-contraction coupling of organ-cultured ileum strips remained essentially normal for at least 1 week after the introduction of antisense ODNs. Cultured, dispersed intestinal smooth muscle cells usually dedifferentiate within 36 hours in culture,⁴⁵ a process that includes the spontaneous downregulation of some membrane-bound proteins. Recently, Rogers et al⁴⁶ reported that strips of organ-cultured canine colonic muscularis externa retained normal ultrastructural, mechanical, and contractile properties when maintained for up to 6 days in their organ-culture system, similar to organ-cultured aortic smooth muscle preparations.⁴⁷ Our organ culture conditions differed from those reported by Rogers et al in that we used a serum-free culture medium. The regular distribution of thin and thick filaments and the normal appearance of the sarcoplasmic reticulum, mitochondria, and nuclei shown in our electron micrographs suggest that over a period of 7 days, organ-cultured rat ileum was not adversely affected by the process of reversible permeabilization, introduction of antisense ODNs, or organ culture itself. The ultrastructure of the rat ileum was, except for the occasional presence of lysosomes, comparable to that of fresh, intact tissue or to those strips fixed immediately after reversible permeabilization, and many of the muscle strips maintained spontaneous contractile activity during the entire course of the experiments. Tension responses to membrane depolarization with high [K⁺] and to carbachol remained similar to those immediately before and after reversible permeabilization. The ODNs or their metabolites had no apparent effect on these parameters, despite a report that metabolites of phosphorothioate ODNs can cause a variety of nonspecific cytotoxic effects.⁷

The phosphorothioate-modified antisense Ang II ODN showed relatively rapid intracellular degradation between 4 and 16 hours after completion of the reversible permeabilization protocol, as estimated by the recovery of internally radiolabeled ODNs. Given a spontaneous reduction in Ang II responsiveness during organ culture and the short (4- to 16-hour) viability of the phosphorothioated ODN in this preparation, it is not surprising that we observed no detectable effect in the antisense ODN–treated tissue.

In ileum sheets containing FITC-labeled antisense ODNs, nuclear fluorescence was detected for at least 7 days by confocal microscopy, even when full-length radiolabeled ODNs were no longer present in cell homogenates. This suggests that fluorescent nucleotide fragments of degraded ODNs were incorporated into nuclear structures, like the hypothesized incorporation of radioactivity from degraded, radiolabeled ODNs into high-molecular-weight structures.⁴³ We could not identify the "ladder pattern" of such ODN fragments in autoradiographs, even after a 1-week exposure, possibly because of their incorporation into DNA or other large macromolecules. In this study, we used only one concentration of ODNs (10 µmol/L) in the reversible permeabilization solutions; whether using higher concentrations of (costly) ODNs would have increased the intracellular concentration of degraded ODNs to more readily detectable levels was not investigated. We conclude that fluorescence microscopy, although useful for tracking the intracellular uptake and distribution of labeled ODNs, does not necessarily reflect their long-term stability.

An unexpected and interesting finding was the nuclear localization of fluorescein-conjugated albumin in reversibly permeabilized but not in control (nonpermeabilized) cells. Cells reversibly permeabilized in the presence of free fluorescein showed distinctly different patterns of labeling. While nuclear targeting of cytosolic proteins is well known, the regulation of such trafficking remains poorly understood. Nuclear pores are permeable to ions^{48 49} and to small protein molecules (<20 kD); however, albumin, lacking a specific nuclear localization signal sequence, is too large to penetrate the nuclear pore complex by diffusion.^{50 51} It is possible that the permeability of the nuclear envelope was enhanced in some fashion during reversible permeabilization. If so, then this method may be useful for introducing other larger molecules, such as transcription regulatory factors, into the nucleus.

The mechanism of reversible plasma membrane permeabilization by ATP in divalent cation–free solutions is not established, although it is thought to involve the binding of ATP^4- to cell surface receptors^{52 53} and the removal of membrane-associated divalent cations.⁵⁴ The resulting increase in membrane permeability is reversible by removal of extracellular ATP and addition of high [Mg²⁺] followed by graded restoration of the physiological concentrations of extracellular Mg²⁺ and Ca²⁺. Reversibly permeabilized tissue sheets were made permeable to phosphorothioate-modified ODNs (M_r4.7 kD), 40-kD and 70-kD fluorescent dextrans, and fluorescein-conjugated albumin (67 kD), whereas in control experiments, fluorescent albumin or dextrans in solutions of HEPES-buffered Krebs' solution at temperatures and times similar to the reversible permeabilization protocol remained extracellular. Larger molecules, including a fluorescent goat anti-rat F(ab')₂ fragment (110 kD) and a fluorescent goat anti-rat IgG (150 kD), were impermeant during reversible permeabilization: the introduction of higher-molecular-weight molecules apparently requires larger and, unfortunately, irreversible pores, such as those produced by ß-escin.⁵⁵ On the basis of these data, we estimate the molecular weight exclusion of the reversible permeabilization technique in ileum to be between 70 and 110 kD, although tertiary and quaternary structures of a protein or its charge-to-mass ratio may also determine whether a particular molecule passes the plasma membrane barrier when this technique is used. In one of three experiments, a plasmid construct (3500 bp; 2.3x10⁶ D) that contained a luciferase reporter under the control of a cytomegalovirus promoter was successfully introduced into reversibly permeabilized ileum sheets: tissue homogenates produced a 60-fold greater light production over homogenates from control tissue loaded with a promoterless luciferase construct (unpublished observations).

We conclude that the feasibility of introducing ODNs into reversibly permeabilized cells in intact smooth muscle bundles, bypassing endosomal uptake, combined with the long-time survival of such preparations in organ culture, provides a highly promising approach for probing the effects of genetic manipulation on the smooth muscle phenotype of excitation-contraction coupling and contractility. Our results also indicate the importance of controls in such studies, both to exclude the nonspecific effects⁸ and to verify the intracellular stability of intact ODNs rather than their fluorescent tags. The latter may be useful, however, for initial localization. The short lifetime of intact intranuclear phosphorothioate ODNs and the spontaneous decrement in Ang II responsiveness in controls may account for the absence of significant phenotypic downregulation of the contractile response to Ang II in our experiments. Pending the development of less readily degradable ODN analogs, we expect to overcome this problem by repeating reversible permeabilization at suitable intervals to reintroduce and maintain the concentration of intact ODNs into organ-cultured smooth muscle. The present methods, however, should have considerable utility for investigations into the biological role of short-lived regulatory molecules in intact, contractile smooth muscle in vitro.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II GTP--S = guanosine 5'-O-(3-thiotriphosphate) ODN = oligodeoxynucleotide SFM = serum-free medium TRITC = tetramethyl rhodamine isothiocyanate

	Acknowledgments

 
This research was supported by Public Health Service Physician Scientist Award 1K11-AR-01871 (Dr Lesh), Public Health Service grant 1PO1-HL-48807 (Dr A.V. Somlyo), and Public Health Service grant PO1-HL-19242 (Drs Owens, A.V. Somlyo, and A.P. Somlyo). We thank Dr Robert Nakamoto for his help with the DNA ligations and Barbara Nordin for preparation of the manuscript. We also wish to acknowledge Mary Alice Spina for her expert technical assistance with the electron micrographs.

Received March 20, 1995; accepted May 5, 1995.

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