Efficacy of a single intravesical treatment with Ad-IFN/Syn 3 is dependent on dose and urine IFN concentration obtained: implications for clinical investigation.

There is a need to improve the treatment of superficial bladder cancer. One area which holds promise is intravesical gene therapy. Recently, studies undertaken by us have shown that marked tumor regression of bladder cancers occurred after two daily intravesical administrations of an adenovirus encoding human interferon alpha (Ad-IFNalpha) using a mouse superficial bladder cancer model in which human bladder tumors are growing. A dose of 1 x 10(11) particles/ml (P/ml) was used along with 1 mg/ml of Syn3, a gene transfer-enhancing agent. Since clinical studies are being planned using this approach, it became critical to determine if one exposure and lower particle number could be equally effective. We report that indeed a single dose of Ad-IFNalpha in Syn3 at doses of 1 x 10(10)-1 x 10(11) P/ml is highly effective in reducing the size of the tumors, whereas 1 x 10(9) P/ml was not. Efficacy was also correlated with the level of IFN produced in the urine after treatment. Based on the results of the present studies, a Phase I trial is being planned for superficial bladder cancer, which will involve a single initial treatment with Ad-IFNalpha/Syn3 and measurement of IFN in the urine over time as an indicator of adequate gene transfer and expression.

Role of alternative splicing of the rat erythropoietin receptor gene in normal and erythroleukemia cells.

An alternatively splicing of the rat erythropoietin receptor (EpoR) gene was identified in normal and erythroleukemia cells by the reverse transcription PCR method. Insertion of a 105 bp fragment at the region corresponding to the extracellular domain of rat EpoR was found. The insert sequence, which encodes additional 21 amino acids, is similar to that previously found in the mouse EpoR gene, however, has an additional 27 bp direct repeat. Due to the presence of a stop codon in the insert, the alternative transcript is translated to a truncated and soluble form of EpoR which is preferentially expressed in liver, spleen, kidney, heart, and bone marrow cells as well as cultured erythroleukemia cells. These findings suggest that alternative splicing of the EpoR gene may play an important role in proliferation and differentiation of rat erythroid cells.

Prostate cancer radiosensitization in vivo with adenovirus-mediated p53 gene therapy.

An adenovirus 5 vector containing wild-type p53 cDNA (Ad5-p53) and a cytomegalovirus promoter was used to generate p53 transgene expression. Control vector (Ad5-pA) contained the poly-adenosine sequence. PC3 cells (2 x 10(6)) were injected s.c. into the legs of nude mice. Treatment with Ad5-p53 was initiated at a tumor volume of 200 mm3. Three intratumoral injections (days 1, 4, and 7) were given with 3 x 10(8) plaque-forming units, followed by 5 Gy pelvic irradiation (day 8) in one fraction using a cobalt-60 source. Tumor volume measurements were obtained every 2 days. LNCaP cells (2 x 10(6)) were injected orthotopically into the prostates of nude mice, and tumor weight was approximated using serum prostate-specific antigen (PSA) obtained from weekly tail vein bleedings. The target PSA for the start of the studies was 5 ng/ml. The intraprostatic injections of Ad5-p53 were done twice (days 1 and 2) and followed by 5 Gy pelvic irradiation on day 3. The PC3 tumor volume growth curves were log transformed and fitted using linear regression. The times (in days) for the tumors to reach 500 mm3 were calculated as 10.7 +/- 0.7 (+/- SE) for the saline control (no virus), 9.8 +/- 2.1 for Ad5-pA, 15.6 +/- 1.6 for Ad5-p53, 14.6 +/- 1.5 radiation therapy (RT; 5 Gy), 14.6 +/- 1.5 for Ad5-pA plus RT, and 31.4 +/- 5.3 for Ad5-p53 plus RT. The Ad5-p53 plus RT times were significantly different from the other groups. An enhancement factor of 3.4 was calculated, indicating supra-additivity. LNCaP tumor growth was determined via weekly serum PSA measurements. Treatment failure was determined using two PSA-based methods; a serum PSA of > 1.5 ng/ml or two rises in PSA during 6 weeks posttreatment. The results were similar using either end point. Treatment with Ad5-p53 plus 5 Gy resulted in significantly fewer PSA failures (<30%), as compared with Ad5-p53 alone (64-73%) and the other controls (approximately 80-100%) These results are also consistent with a supra-additive inhibition of tumor growth. Tumor growth in vivo was inhibited supra-additively when p53null and p53wildtype prostate tumors were treated with Ad5-p53 and 5 Gy radiation.

Adenoviral-mediated p53 transgene expression sensitizes both wild-type and null p53 prostate cancer cells in vitro to radiation.

PURPOSE/OBJECTIVE: The effect of adenoviral-mediated p53 transgene expression on the radiation response of two human prostate cancer cell lines, the p53(wild-type) LNCaP and p53(null) PC3 lines, was examined. The objective was to determine if this vector sensitizes cells to radiation independently of their p53 status. METHODS AND MATERIALS: A recombinant adenovirus-5 vector (RPR/INGN 201, Introgen Therapeutics, Houston, TX) containing a CMV promoter and wild-type p53-cDNA (Ad5-p53) was used to facilitate p53 transgene expression. A multiplicity of infection (MOI) of 10-40 viral particles per cell was used, based on Ad5/CMV/lacz infection and staining for the beta-galactosidase reporter gene product. Clonogenic assays were performed to evaluate the degree of sensitization to radiation of viral-transduced cells compared with irradiated nontransduced controls. The relative efficacy of these treatments to induce apoptotic cell death was determined using the TUNEL assay. RESULTS: The delivery of Ad5-p53 (10 MOI) reduced control plating efficiency from 36.5% to 0.86% in the LNCaP cell line and from 75.1% to 4.1% in the PC3 cell line. After correcting for the effect of Ad5-p53 on plating efficiency, the surviving fraction after 2 Gy (SF2) of gamma-irradiation was reduced over 2.5-fold, from 0.187 to 0.072, with transgene p53 expression in the LNCaP cell line. Surviving fraction after 4 Gy (SF4) was reduced over 4.5-fold, from 0.014 to 0.003, after Ad5-p53 treatment. In the PC3 cell line, Ad5-p53 (40 MOI) reduced SF2 over 1.9-fold from 0.708 to 0.367, and SF4 over 6-fold from 0.335 to 0.056. In both the LNCaP and PC3 cell lines, the combination of Ad5-p53 plus radiation (2 Gy) resulted in supra-additive apoptosis (approximately 20% for LNCaP and approximately 15% for PC3 at 50 MOI), above that seen from the addition of the controls; control vector Ad5-pA plus RT (0.15% for LNCaP and 1.44% for PC3), Ad5-p53 alone (28.6% for LNCaP and 21.7% for PC3), RT alone (0% for LNCaP and 0.23% for PC3), or Ad5-pA alone (0.1% for LNCaP and 0.29% for PC3). CONCLUSION: The clonogenic survival and apoptosis data demonstrate that p53 transgene expression sensitizes human prostate adenocarcinoma cells in vitro to irradiation. As this effect was observed in both the p53(wild-type) LNCaP and p53(null) PC3 lines, radiosensitization was independent of p53 status.

The early supra-additive apoptotic response of R3327-G prostate tumors to androgen ablation and radiation is not sustained with multiple fractions.

PURPOSE: The treatment of R3327-G tumor-bearing rats with androgen ablation (AA) via castration results in a supra-additive increase in apoptosis when 2-8 Gy gamma-irradiation (RT) is given as a single dose 3-14 days afterwards. We report here the dose response and effect of multiple fractions on this supra-additive apoptotic response. MATERIALS AND METHODS: Dunning R3327-G tumors were grown in the flanks of Copenhagen rats and the experiments were initiated at a tumor volume of 1.0-1.5 cc. Androgen ablation was achieved by castration 3 days prior to gamma-irradiation. Apoptosis was measured with a terminal deoxynucleotidyl transferase dUTP-biotin nick end-labeling assay 6-h after RT, unless otherwise specified. RESULTS: The dose response of the supra-additive apoptotic response was assessed by irradiating castrated animals with single doses of 2, 4, 8, or 16 Gy (n = 5 per group); tumor cell apoptosis at 6-h following irradiation was 2.4%+/-0.7% (+/- SEM), 4.2%+/-0.8%, 6.5%+/-1.4%, and 1.6%+/-0.3%, respectively. The RT only and AA only controls had < 1% apoptosis. The effect of fractionated RT on apoptosis was investigated to determine if the supra-additive apoptotic response was sustained with repeated 2-8 Gy fractions. When tumor-bearing animals were treated with repeated daily 2-Gy fractions, there was a reduction in the level of the supra-additive apoptotic response. After five 2-Gy fractions at 24-h intervals, apoptosis in the combined treated tumors was at levels seen in the AA controls. This raised the possibility that more than 24 h are required for recovery of the high supra-additive apoptotic levels seen after one fraction. When the interfraction interval was extended to 96 h, there was no significant increase in apoptosis over the additive effect of AA and RT. Although there was a decline in supra-additive apoptosis with repeated fractions, a dose response for tumor growth delay was evident for RT alone using 2.5-Gy fractions. Moreover, the combination of AA + fractionated RT resulted in a supra-additive enhancement in tumor growth delay to 5 cc. CONCLUSION: The early supra-additive apoptotic response from AA and single fraction radiation is not seen at high single fraction doses and is not sustained with repeated fractions. Therefore, the classical apoptotic response that occurs within 24 h of irradiation is not likely to be the main mechanism responsible for any clinical benefit seen with this combination.

Lack of prostate cancer radiosensitization by androgen deprivation.

PURPOSE: The majority of clinical trials have shown that high-grade prostate cancer patients treated with androgen deprivation (AD) plus radiation (RT) have a survival advantage over those treated with RT alone. One possible mechanism for such a favorable interaction is that AD sensitizes cells to radiation. Animal model studies have provided suggestive evidence that AD sensitizes cells to radiation, but this mechanism is difficult to confirm conclusively in vivo. This question was investigated in LNCaP cells grown in vitro. METHODS AND MATERIALS: LNCaP cells were cultured in vitro in Dulbecco's modified Eagle's medium (DMEM)-F12 medium, containing 10% fetal bovine serum (complete medium [CM]). AD was achieved by culture in charcoal-stripped serum (SS)-containing medium. Replacement of androgen was done by adding the synthetic androgen R1881 at 1 x 10(-10) M to SS. Apoptosis was measured with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Clonogenic survival was used to determine overall cell death, and the results were corrected for differences in plating efficiency from the various growth conditions. RESULTS: LNCaP cells were grown in CM, SS, or SS + R1881 medium, and cell counts obtained at 3, 4, and 5 days. Cell number increased exponentially in CM, whereas no increase in cell number was observed in SS medium. Cell counts from growth in SS + R1881 were intermediate between these extremes. Apoptosis was measured to determine if the combination of AD + RT in vitro resulted in supra-additive cell death, as has been previously described in an in vivo model system. The cells were cultured for 3 days before RT and apoptosis quantified 24 h after RT. There was a consistent supra-additive increase in apoptosis in cells exposed to AD + RT (2 or 8 Gy), as compared to either treatment given individually. In contrast, significant radiosensitization by AD was not observed by clonogenic survival even when the conditions of AD were varied. No radiosensitization was observed upon incubation in SS medium for 3, 4, or 5 days before RT, or extending AD after RT for 6 h before plating or 24 h after plating. CONCLUSION: The results show that in LNCaP prostate tumor cells supra-additive apoptosis does not translate into radiosensitization by clonogenic survival. Because clonogenic survival is a measure of overall cell death, either the level of apoptosis is too small a component of overall cell death or the increases in apoptosis occurred in a subpopulation that would have been killed by other mechanisms. Although the findings indicate that AD does not act by sensitizing prostate cancer cells to RT, the additive cell death and growth inhibitory effects of AD + RT are clinically meaningful.

Carbachol-induced contraction in the circular muscle of guinea-pig stomach in calcium-free solution.

In the circular muscle of the fundic part of the guinea-pig stomach, a small tonic contraction could be repeatedly produced by carbachol in Ca-free solution containing 2 mM EGTA. The carbachol-induced response was gradually increased during prolonged exposure to Ca-free solution for 50 h, whereas a short treatment with 0.1-0.2 mM Ca suppressed the subsequent carbachol response in Ca-free solution. The response was not essentially modified by increasing the external K+ concentration to 40 mM. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) and trifluoperazine selectively suppressed the carbachol response in the presence of Ca (0.05 mM) and the contraction induced by Ca (0.1 mM), but they had little effect on the response to carbachol in Ca-free solution at a concentration of less than 10 microM. A vasodilator agent, N-(2-guanidinoethyl)-5-isoquinoline sulphonamide (HA-1004), inhibited the carbachol response both in the presence and absence of Ca, as well as the Ca-induced contraction, to a similar extent, provided that the external Ca concentration was less than 0.1 mM. These results led us to propose that the contraction evoked by carbachol in the absence of external Ca is mediated by a process independent of the Ca-calmodulin system.

The response of non-pregnant rat myometrium to oxytocin in Ca-free solution.

The contractile response of the longitudinal muscle of non-pregnant rat myometrium to oxytocin (0.2-20 nM) consisted of a phasic and a tonic component. Ca-removal abolished the phasic component but a tonic contraction could be evoked without reduction of amplitude for 50 h. Exceptionally, the tonic contraction also disappeared gradually in Ca-free medium containing 2 mM EGTA. When oxytocin was repeatedly applied in the absence of Ca, the response became at first progressively larger before reaching a steady state. Transient addition of Ca to the medium reduced the size of the subsequent oxytocin contraction. In Ca-free medium, the tissue lost Ca slowly, but it still contained 40 mumol kg-1 after 6 h and roughly 1 mumol kg-1 wet weight after 24 h exposure. 45Ca efflux was marginally increased by oxytocin (20 nM). Caffeine (5-30 mM) produced no contraction, but slightly reduced the resting tension and strongly inhibited the oxytocin response both in the presence and in the absence of Ca. Caffeine also blocked the contraction induced by Ca added to Ca-free 40 mM K solution. However, pretreatment with caffeine (30 mM) had no effect on the following oxytocin response. A calmodulin antagonist, trifluoperazine (1-10 microM) suppressed strongly the Ca-induced contraction, but had only a weak effect on the oxytocin response in Ca-free medium. Chlorpromazine (10-100 microM) and fluphenazine (10-30 microM) had similar effects. A different type of antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) (0.1 mM) almost completely blocked responses to both oxytocin and to Ca, but recovery of the Ca-induced contraction was much better than that of the oxytocin response in Ca-free solution. 6 Since no evidence was found for intracellular Ca release by oxytocin, and as there were several differences between the effects of calmodulin antagonists on the oxytocin response and on the Ca-induced response of similar size, the possibility remains that some Ca-independent process is involved in the contractile response to oxytocin observed in Ca-free solution.

Alternative splicing of the erythropoietin receptor gene correlates with erythroid differentiation in rat hematopoietic and leukemic cells.

An alternative splicing of the rat erythropoietin receptor (EpoR) gene was identified in normal and erythroleukemia cells. A 105 bp insert was found at a region corresponding to the extracellular domain of EpoR. The alternative transcript was translated to a soluble EpoR (EpoR-S) expressed in spleen, bone marrow, and cultured erythroleukemia cells in addition to the full-length EpoR (EpoR-F). One of the rat erythroleukemia sublines, K4DT, which partially lost erythroid phenotypes and manifested monocyte/macrophage characteristics also lacked EpoR-S expression. Thus, expression of EpoR-S may play an important role in differentiation of rat erythroid cells.

Involvement of lipid peroxidation in necrosis of skin flaps and its suppression by ellagic acid.

To evaluate the pathogenesis of lipid peroxidation in skin-flap necrosis and to select a novel herbal antioxidant to suppress lipid peroxidation and salvage the flaps, in vitro and in vivo experiments were instituted. In vitro studies revealed (1) the potentiality of the cutaneous microsomal system (vesicular fragment of endoplasmic reticulum) to generate oxyradicals by FeCl3 (oxidative agent), since NADPH-dependent lipid peroxidation was elevated time-dependently, (2) suppression of microsomal lipid peroxidation by herbal antioxidants (dose- and time-dependently), further supporting the theory of oxyradical-induced lipid peroxidation in the skin, and (3) that ellagic acid showed the strongest response, with curcumin, chlorogenic acid, and alpha-tocopherol (tocopherol) being moderate, and ferulic acid and gallic acid remaining weakest. Thus ellagic acid, curcumin, chlorogenic acid, and tocopherol at doses of 10, 60, 80 and 100 microM (twice I50, the dose which could inhibit lipid peroxidation by 50 percent) were chosen for in vivo assessments, respectively. In vivo studies were performed using rat back skin random flaps (70 x 15 mm and based anteriorly) and circular island flaps (20 mm in diameter and raised on superficial epigastric vessels). Control flaps were painted with a Tris-ethanol solution, and test flaps were painted with either ellagic acid, curcumin, chlorogenic acid, or tocopherol (above-mentioned doses per 250 microliters of Tris-ethanol per 300 mm2 of flap surface 1 hour before the operation and once a day for 3 postoperative days). Doses, frequency, and period of drug application were based on in vitro and in vivo pilot experiments. The results were as follows: (1) a direct and time-dependent relation was noticed between lipid peroxide levels and the rate of necrosis in both types of flap; (2) time-dependent elevation of lipid peroxide levels of skin, subcutaneous fat, and exudate of island flaps during ischemia and those of skin and subdermal fat after reperfusion indicated pre- and post-reflow states of lipid peroxidation rather than the original conception of merely reperfusion state; and (3) in good agreement with the results of in vitro experiments, ellagic acid exerted the strongest effect to suppress lipid peroxide levels of skin and to augment the viability of random flaps more than that of island flaps.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible contributions of mastocytosis, apoptosis, and hydrolysis in pathophysiology of randomized skin flaps in humans and guinea pigs.

To understand better the pathophysiology of random skin flaps, randomized skin flaps of human (3 cases) and guinea pig (53 cases) were investigated. Proximal (normal), proximomedial (viable), mediodistal (between viable and necrotic parts), and distal (necrosis) locations of the skin flaps were biopsied. Lipid peroxidase, hydrolytic enzymes of cytosol (Ca(2+)-dependent cysteine protease: calpain), and lysosome (acid phosphatase) of skin were used as markers. Measurements were taken of the flap blood flow; the numbers of capillaries, postcapillary venules, pericapillary arterioles, leukocytes, and mast cells per unit square of dermis. Apoptotic cells were identified by specific staining. Flaps were sampled at postoperative weeks 1 and 3 (human) and hours 1 and 6, and days 1 to 7 (guinea pig). The values for normal skin were regarded as the control. Obstruction (by leukocytes) of venous microvessels, rather than arterial microvessels, was the major cause of temporary hypoxia in the proximomedial location, constant hypoxia (venous stasis) in the mediodistal location, and ischemia in the distal location. Increases in the number of mast cells (mastocytosis) and microvessels (angiogenesis) were significant only in the viable parts of the flaps. This phenomenon and the rate of blood flow increased with time in viable locations (guinea pig). Epidermal necrosis, dermal fibrosis, and apoptosis were evident mostly in the mediodistal location. Elevated levels of leukocytes, lipid peroxidase, acid phosphatase, and calpain, combined with necrotic changes, were seen mostly in the distal skin location. There is a strong possibility that the following factors are involved: lipid piroxidation and hydrolysis in necrosis of the distal flap location after ischemia; constant hypoxia in fibrosis and apoptosis in the mediodistal location; and initial or temporary hypoxia in mastocytosis-induced angiogenesis in the viable location. The results presented here indicate that guidelines for further investigations include combined suppression of leukotaxis, lipid peroxidase, and hydrolysis, or the application of mast cell growth factors in an effort to salvage the flap maximally.

Early RB94-produced cytotoxicity in cancer cells is independent of caspase activation or 50 kb DNA fragmentation.

RB94, which lacks the N-terminal 112 amino-acid residues of the full-length retinoblastoma protein (RB110) is a more potent inhibitor of cancer cell growth than RB110, being cytotoxic to all cancer cell lines studied, independent of their genetic abnormalities. Although we initially thought RB94-induced cell death was caspase-dependent, such caspase activation now appears to be a late event. Cells that remained attached 48 h after transduction with Ad-RB94 showed, among other changes, nuclear enlargement, peripheral nuclear chromatin condensation and often micronucleation. In addition, the cells were TdT-mediated dUTP nick end labeling (TUNEL) positive but showed no cleavage of caspase 3 or 9. Only after the cells detached was cleavage of both caspase 3 and 9 observed. These TUNEL-positive cells showed neither cytochrome c mitochondrial translocation usually found in typical apoptotic cells nor DNA laddering indicative of oligonucleosomal DNA fragmentation. In addition, although 50 kb DNA fragmentation was produced in these TUNEL-positive cells, which was dependent on apoptosis-inducing factor (AIF), inhibiting this fragmentation by siAIF did not inhibit TUNEL formation or cytotoxicity. As RB94 will soon be used for gene therapy further understanding the molecular basis of these early changes in killing cancer cells is one of our particularly important present goals.

Mechanical response to noradrenaline in calcium-free solution in the rat vas deferens.

Mechanical responses to noradrenaline (NA) were investigated in the rat vas deferens exposed to Ca-free solution containing 0.5 mM-EGTA. A tonic response was produced in Ca-free solution at the epididymal portion, while almost no response could be observed at the prostatic portion. In most experiments NA (10(-4) M) was applied for 4 min, every 20 min. The absolute tension development in Ca-free solution was usually 60-80% of the control tonic response in the presence of 2.4 mM-Ca. The response could be produced repeatedly, even after exposure to Ca-free solution for more than 20 hr, without a significant decrease. During the first hour of exposure to Ca-free solution, the rate of rise and the magnitude of the NA contraction increased and then remained constant, though the relaxation became slow. Transient treatment with 2.4 mM-Ca slightly suppressed the subsequent NA response in Ca-free solution. Similarly, the NA response was smaller during readmission of 0.2-0.5 mM-Ca than that obtained before Ca readmission. A high concentration of verapamil (2 X 10(-4) M) reversibly reduced the NA response by about 70% after 30 min. Theophylline (10 mM) and dibutyryl cyclic AMP (10(-4) M) also reversibly suppressed the NA response, the suppression being about 80%. None of these substances produced a tension change by themselves. The suppressing effect may be mediated via an increase of intracellular cyclic AMP which reduces phosphorylation of myosin. Caffeine (10 mM) and dibutyryl cyclic GMP (10(-4) M) had similar but much weaker effects than theophylline and dibutyryl cyclic AMP. A calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) slowly reduced the NA response. The block was nearly complete after 30 min treatment with 3 X 10(-4) M-W-7, and the recovery was very poor after prolonged exposure. This effect of W-7, which is the same in the presence and absence of Ca, suggests that a Ca-calmodulin reaction is involved in the NA response in Ca-free solution. Fluoride at a concentration higher than 3 mM increased the muscle tone in the absence of external Ca, and transiently potentiated the NA response. In the presence of F-, the relaxation of the NA response was incomplete and the muscle tone increased stepwise after each NA application. When the muscle tone became higher than the NA response in the absence of F-, the NA response was abolished. The action of several metabolic inhibitors (2,4-dinitrophenol, carbonylcyanide chlorophenyl hydrazone, NaCN, monoiodoacetate) was similar to that of F-, suggesting that they release Ca from mitochondria, causing tension development. The observations are consistent with the hypothesis that the contraction of the vas deferens caused by NA in the absence of external Ca depends on the availability of intracellular Ca, stored in mitochondria and released by NA.

Contraction of smooth muscle in Ca-free solution.

The tonic contractions which are extremely resistant to removal of the external Ca were investigated in the rat vas deferens and myometrium. Both the noradrenaline response in the vas deferens and the oxytocin response in the myometrium could be repeatedly produced without appreciable diminution in Ca-free solution for more than 24 hrs. On the other hand, the tissue Ca content decreased exponentially after Ca-removal with a half time of 130-180 min. When Ca was readmitted, no indication of the early transient contraction was observed in the subsequent response in Ca-free solution, but the response was reduced compared with the response before Ca readmission. Verapamil suppressed the response in the presence of Ca, while it had very weak inhibitory effect even at 10 microM. Calmodulin antagonists of phenothiazine derivatives had a strong inhibitory effect on Ca-induced contractions, whereas they had very weak effects on the receptor-mediated contraction in Ca-free solution. Another calmodulin antagonist, W-7 suppressed both Ca-induced contraction and the contractions independent of external Ca. HA-1004, a vasodilator which has a structure similar to W-7, reduced the receptor-mediated contraction in Ca-free solution without much effect on Ca-induced contractions. These results may suggest that the receptor-mediated contractions resistant to Ca-removal are caused by some process without a contribution of the Ca-calmodulin system.

Effects of glucose removal and readmission on potassium contracture in the guinea-pig taenia coli.

The effects of removal and readmission of substrates on the K contracture were investigated in the guinea-pig taenia coli. When, after exposure to excess K in Ca-free and glucose-free medium, the readmission and removal of 2.4 mM-Ca were repeated at regular intervals, the Ca-induced contractions decreased progressively. The decrease was more marked in the late than in the early part of the tension response. The rate of O2 consumption decreased when the normal medium was replaced by glucose-free, Ca-free, excess-K solution, but substantially recovered following Ca readmission. ATP and creatine phosphate contents decreased during the Ca-induced contraction, but recovered partially during the subsequent relaxation in Ca-free solution. The effects of glucose removal were rapidly reversed when glucose or beta-hydroxybutyrate (beta-HB) were readmitted to the bathing solution. In the absence of Ca, readmission and removal of the substrates produced an insignificant change in O2 consumption, but the next Ca contraction was potentiated, the effect being stronger with glucose than beta-HB. When the tonic contraction evoked by 2.4 mM-Ca readmission had been abolished in glucose-free, high-K solution, a rise of the external Ca concentration to 10 mM, or 5 microM-carbachol, still produced a transient contraction. This suggests that the tonic contraction has disappeared partially because of diminished Ca influx. In glycogen-depleted preparations, the depolarization caused by carbachol, or by 20 mM-K, was increased and spike discharge initiated when glucose was readmitted.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term follow-up study of artificial dermis composed of outer silicone layer and inner collagen sponge.

In the oriental population including the Japanese, donor-site hypertrophy is more pronounced than in Caucasians. To solve the problem of donor-site morbidity and to ensure graft 'take', we started the second-stage procedure of a thin split thickness skin graft (STSG) onto acellular 'bilayer artificial skin', or 'artificial dermis'. Since reporting the original version of the material (OV), a revised version (RV) and the present version (PV, Pelnac) have been developed in stages to eliminate inconveniences associated with its use and to reduce the primary cost of manufacture. We have now used our materials, consisting of OV, RV and PV, on 52 skin defects in 41 patients. STSG took almost perfectly in all patients. The long-term results of these three materials were investigated in 20 patients who had been followed up for more than 2 years, excluding three patients whose donor sites had been directly closed. The longest and the mean follow-up periods of these patients were 12 years 5 months and 6 years 10 months, respectively. At the grafted sites, wrinkles caused by shrinkage, partial depigmentation and hypertrophy were observed in five (25%), one (5%) and one (5%) of the 20 patients, respectively. At the donor sites, slight unsightliness was observed in five (25%) of the 20 patients. Excellent or good results were obtained in 18 (90%) of the 20 patients in comprehensive evaluation. There were no significant differences in the long-term follow-up evaluations among these materials. In conclusion, the long-term postoperative appearance of the STSG site was good though a very thin (approximately 0.2mm) STSG is used; scarring of the donor site was minimal and it was possible to take repeated skin grafts from the same donor site.