Contraction and intracellular calcium-ion elevation of cultured human aortic smooth muscle cells by endothelin-1, vasoactive intestinal contractor (VIC) and the derivatives

Effects of endothelin (ET) family peptides and their derivatives on cellular contraction and calcium-ion level were examined by using cultured human vascular smooth muscle cells (VSM). Contraction of cultured human VSM, isolated from human fetal aortic segments, was induced within 1 min after the treatment with ET-1 (100 nM) as seen in the changes of cytosolic calcium-ion localization. In parallel with the cell contraction, cytosolic calcium-ion level in the human VSM increased very rapidly and then dropped with some oscillation as determined by Anchorage Cell Analyzing System. It was noted that transient calcium-ion mobilization rather than sustained calcium-ion influx was significant in the contraction of cultured human VSM. Vasoactive intestinal contractor (VIC), three amino acids different from ET-1, had less activity in increase of intracellular calcium-ion level and in percent of response cells than ET-1, ET-2, and VIC-S4L6 (one amino acid different from ET-1). EC50 of ET-1, VIC-S4L6, ET-2, and VIC were 0.5 nM, 0.6 nM, 2.0 nM, and 20 nM, respectively. VIC-like peptide (VIC-LP), 16 amino acids fragment of VIC precursor protein, had no effect with a single administration of up to 10 microM. However, the increase in calcium-ion level by VIC was suppressed with a prior treatment of cells with high concentration (10 microM) of VIC-LP. The establishment of cultured human VSM for the simultaneous examination of the contraction and calcium-ion level will provide a new system to study signal transduction of vasocontractor peptides.     

Protein kinase C alterations in aortic vascular smooth muscle cells from rats with cirrhosis

It was found that the frequency of extreme situations (traumas, deaths, sudden diseases) in miners working in Spitsbergen mines (74 degree N) correlates well with heliogeomagnetic activity (local magnetic disturbances, solar proton flashes). It was shown that in winter, both an enhanced and an extremely low level of magnetic activity can affect the occurrence of extreme situations. The results obtained can be used for predicting and reducing the frequency of extreme events in the zone of the polar cap during geomagnetic disturbances.     

Effect of isoflurane on endothelin-1 mediated airway smooth muscle contraction

Endothelin-1 (ET-1) is an endogenously produced 21 amino acid peptide that can cause significant airway smooth muscle (ASM) constriction. Volatile anesthetics e.g. isoflurane, can attenuate ASM constriction. This investigation was undertaken to study the direct effects of isoflurane, at a clinically relevant dose, on endothelin-induced constriction in rat trachea. Five Sprague-Dawley female rats (weight 325-350 g) were euthanized. Four 2-3 mm rings were excised from the mid portion of the trachea, attached to a force transducer for isometric measurement, and were then suspended in jacketed baths containing 37 degreesC KH solution, bubbled with 95% O2 and 5% CO2. Carbachol dose-response curves (10(-8)-10(-4)M) were obtained to establish the maximal contractility (Cmax) for each tracheal ring. Carbachol was then washed out of the bath solution until the tissue tension returned to the baseline. Subsequently, ET-1 dose response curves (3-200 nM) were generated in the absence (ET-1, control) and in the presence of 2% isoflurane. Our data suggests that isoflurane at 2% concentration, attenuated ET-1 induced tracheal contraction at 100 and 200 nM concentrations by 20%. This is the first demonstration indicating that, at a clinically relevant dose, isoflurane depresses ET-1 induced ASM constriction.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

Hyperproliferation of aortic smooth muscle cells and fibroblasts from young SHR rats is not shared by endothelial cells

1. To study the hypertensive genotypic influence on growth kinetics of the three aortic wall cell types. 2. Using young spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats weighing 160-180 g, we compared the proliferative properties of endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts that were isolated from the thoracic aorta of each strain and cultured. Growth-arrested cells were exposed to P < -thymidine after stimulation with 150 micrograms/mL endothelial cell growth supplement. Proliferation assays were performed by cell seeding on decellularized aortic explants and cell counting 2, 4, 5, 6 and 7 days after seeding. The influence of SMC from SHR on the growth kinetics of EC was evaluated by co-cultures in transwell systems. 3. After stimulation, SMC from SHR exhibited a greater P < -thymidine incorporation rate than those from WKY rats (ratios over controls: 3.90 +/- 0.48 [7] vs 1.85 +/- 0.25 [7] respectively, P < 0.05). This was also true for adventitial SHR fibroblasts: (13.1 +/- 0.6 [6] vs 9.9 +/- 1.0 [6] WKY P < 0.05). On the contrary, there was no difference in the P < -thymidine incorporation rates between EC of each strain, regardless of the passage and the time considered. Cell proliferation on matrix explants confirmed the hyperproliferation of SMC and fibroblasts from SHR, while EC of each strain proliferated equally. Smooth muscle cells from SHR did not influence the growth kinetics of EC in co-culture and vice versa. 4. The changes in growth patterns of aortic cells isolated from young prehypertensive SHR seem to be restricted to SMC and fibroblasts.     

Effect of pressure on cultured smooth muscle cells

Recent studies indicate that smooth muscle cell (SMC) growth and morphology can be modulated by repetitive strain. However, there is very little known about the influence of pressure, without an associated cell stretch, on SMC phenotype. To study this, cultured bovine aortic SMC were grown on a rigid surface and placed in a custom-designed plexiglass pressure chamber with a carefully regulated 5% CO2/air environment. SMC were exposed to either atmospheric, 105 mm Hg or 120/90 mm Hg pressure at a frequency of 60 cycles/min (0.5 s systole, 0.5 s diastole). SMC number was determined on days 1, 3, 5, 7 and 9. SMC exposed to pressure were more elongated and displayed a significant increase in cell number by day 5 which persisted until day 9. Lactate dehydrogenase (LDH) in the conditioned media, an index of cytotoxicity, was not different between the groups at each time point. There was also no difference in pH or pCO2 of the media of SMC in any group. This is the first report of the effects of increased static and pulsatile pressure on SMC in vitro and indicates an increased proliferative rate. We hypothesize that the systemic pressure that the blood vessel is exposed to in vivo may have a significant regulatory influence on the phenotype of the smooth muscle cells which may affect the SMC response to injury or stimuli.     

Developmental regulation of perlecan gene expression in aortic smooth muscle cells

The effect of environmental temperatures on immune competence was investigated in carp which were subjected to changes in water temperature. The activity of non-specific cytotoxic cells (NCC) against P815 target cells, and the anti-DNP antibody response were evaluated until day 56 after transfer. Low environmental temperature (12 +/- 0.5 degrees C) enhanced NCC activity and decreased antibody production. In contrast a high environmental temperature (28 +/- 0.5 degrees C) was without effect on these parameters when compared to the standard temperature (20 +/- 0.5 degrees C). The results showed a maximum effect of low environmental temperature on day 28 and an adaptation in these immune responses 56 days following transfer. Collectively, the results indicated that non-specific immunity tends to offset specific immune suppression at low environmental temperatures. To determine the mechanism(s) by which environmental temperature affects cellular immune function, membrane fluidity measurements and sialic acid titration, as well as stress assessment by plasma cortisol measurement, were determined on day 28. Taken together, the results revealed a direct effect of temperature on cellular immune function which is modulated by membrane fluidity and sugar concentration and not by stress induction.     

Altered growth and lack of responsiveness to angiotensin II in aortic vascular smooth muscle cells from cirrhotic rats

BACKGROUND & AIMS: In cirrhosis, increased amounts of circulating hormones such as angiotensin II may induce vascular tone changes and alter vascular smooth muscle cell (VSMC) function and growth. The aim of this study was to investigate the growth of aortic VSMCs from cirrhotic rats with or without the addition of angiotensin II and to determine whether angiotensin II binding was preserved in cirrhotic VSMCs. METHODS: Cirrhosis was induced by bile duct ligation. Cell growth was studied in cultured aortic VSMCs at passage levels between 4 and 16 by determining cell number and protein synthesis. RESULTS: Proliferation rates of cirrhotic VSMCs were lower than those of control VSMCs. The addition of angiotensin II to control VSMCs caused an increase in cell proliferation and protein synthesis. This increase was not observed in cirrhotic cells. There were more angiotensin II receptors in cirrhotic than in control VSMCs, but no significant changes in affinities were found. Angiotensin II-stimulated protein synthesis was dependent on protein kinase C activity and increased intracellular Ca2+ concentrations. CONCLUSIONS: This study shows abnormalities in growth characteristics and responsiveness to angiotensin II of cultured aortic VSMCs from rats with cirrhosis.     

Nitric oxide inhibits Oct-1 DNA binding activity in cultured vascular smooth muscle cells

In this paper, the authors model the nonmonotonic relation between body mass index (BMI) (weight (kg)/height2 (m2)) and mortality in 13,242 black and white participants in the NHANES I Epidemiologic Follow-up Study in order to estimate the BMI at which minimum mortality occurs. The BMI of minimum mortality was 27.1 for black men (95% confidence interval (CI) 24.8-29.4), 26.8 for black women (95% CI 24.7-28.9), 24.8 for white men (95% CI 23.8-25.9), and 24.3 for white women (95% CI 23.3-25.4). Each confidence interval included the group average. Analyses conducted by smoking status and after exclusion of persons with baseline illness and persons who died during the first 4 years of follow-up led to virtually identical estimates. The authors determined the range of values over which risk of all-cause mortality would increase no more than 20% in comparison with the minimum. This interval was nine BMI units wide, and it included 70% of the population. These results were confirmed by parallel analyses using quantiles. The model used allowed the estimation of parameters in the BMI-mortality relation. The resulting empirical findings from each of four race/sex groups, which are representative of the US population, demonstrate a wide range of BMIs consistent with minimum mortality and do not suggest that the optimal BMI is at the lower end of the distribution for any subgroup.     

The cytoskeleton in skeletal, cardiac and smooth muscle cells

The muscle cell cytoskeleton consists of proteins or structures whose primary function is to link, anchor or tether structural components inside the cell. Two important attributes of the cytoskeleton are strength of the various attachments and flexibility to accommodate the changes in cell geometry that occur during contraction. In striated muscle cells, extramyofibrillar and intramyofibrillar domains of the cytoskeleton have been identified. Evidence of the extramyofibrillar cytoskeleton is seen at the cytoplasmic face of the sarcolemma in striated muscle where vinculin- and dystrophin-rich costameres adjacent to sarcomeric Z lines anchor intermediate filaments that span from peripheral myofibrils to the sarcolemma. Intermediate filaments also link Z lines of adjacent myofibrils and may, in some muscles, link successive Z lines within a myofibril at the surface of the myofibril. The intramyofibrillar cytoskeletal domain includes elastic titin filaments from adjacent sarcomeres that are anchored in the Z line and continue through the M line at the center of the sarcomere; inelastic nebulin filaments also anchored in the Z line and co-extensible with thin filaments; the Z line, which also anchors thin filaments from adjacent sarcomeres; and the M line, which forms bridges between the centers of adjacent thick filaments. In smooth muscle, the cytoskeleton includes adherens junctions at the cytoplasmic face of the sarcolemma, which anchor beta-actin filaments and intermediate filaments of the cytoskeleton, and dense bodies in the cytoplasm, which also anchor actin filaments and intermediate filaments and which may be the interface between cytoskeletal and contractile elements.     

Relationship between blood pressure and smooth muscle tone in aortae of hypertensive rats: roles of [Ca2+]

Spontaneously developed tension (active tone) and intracellular Ca2+ level of aortae from spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), malignant SHRSP (M-SHRSP) and control Wistar Kyoto rats (WKY) were compared. Systolic blood pressure of WKY, SHR, SHRSP and M-SHRSP was 130 mmHg, 200 mmHg, 250 mmHg and 260 mmHg, respectively. Preparations from all strains of spontaneously hypertensive rats exhibited active tone which was abolished by the removal of extracellular Ca2+ or by the application of verapamil. The active tone was greater in the order of aortae from SHR < SHRSP < M-SHRSP. Intracellular Ca2+ level measured by Fura-2 method decreased by the removal of extracellular Ca2+. The degree of the decrease was greater as the blood pressure of the rats increased, indicating the greater elevation of intracellular Ca2+ level in the pressure of extracellular Ca2+. A correlation was obtained between the active tone, intracellular Ca2+ level and blood pressure. Thus, it was demonstrated that the development of the active tone is brought about by the changes in Ca2+ influx of smooth muscle cell membrane and the degree of the change is positively related to the degree of hypertension.     

A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells

OBJECTIVE: To evaluate the predictive value of clinical variables for the finding of a positive minor salivary gland biopsy (focus score > or = 2) in patients investigated for Sj?gren's syndrome (SS). METHODS: One hundred twenty-one patients with sicca symptoms were referred to a multidisciplinary SS clinic in a tertiary hospital. Each patient was evaluated on protocol and labial salivary gland (LSG) biopsy was obtained. Using the San Diego criteria as a model, patient data were subjected to a cross sectional analysis on an algorithm to determine when the LSG biopsy would be most useful for determining the diagnosis of SS in clinical practice. RESULTS: Eighty-four patients had sufficient data to be included in the study. Forty patients had LSG biopsy with focus score < 2 and 44 had focus score > or = 2. Twenty-three patients had objective evidence of sicca and positive serology according to criteria standards. Eighteen of these had a positive biopsy (78%). The remaining 5 patients had many extraglandular features suggestive of SS, and the biopsies appeared to add little practical information. Patients with incomplete criteria for sicca could be diagnosed as possible SS (3 of 4 criteria) with a positive biopsy in 14 of 18 cases. The finding of anti-Ro or anti-La positivity in patients with incomplete criteria for sicca predicted a positive LSG biopsy in 85.7% of such cases. Patients with incomplete sicca and negative anti-Ro and anti-La had a negative LSG biopsy in 82% of cases. CONCLUSION: The LSG biopsy is most necessary in patients who have partial San Diego criteria for sicca and positive anti-Ro or anti-La antibody. Where SS is not reasonably suspected, or where the diagnosis is clinically obvious, the LSG biopsy adds little useful clinical information.     

Synthesis and removal of different cholesterol esters by aortic smooth muscle cells in culture

The incorporation of 3H-labelled oleic acid and of 14C-labelled linoleic acid into phospholipid, triglyceride and cholesterol ester in smooth muscle cells grown in incubation medium supplemented with either 5% normal or 5% hyperlipemic serum has been studied. Both fatty acids were incorporated into cholesterol esters to a greater extent when cells grown in incubation medium containing hyperlipemic serum. Oleic acid was incorporated into cholesterol esters in preference to linoleic acid. The addition of hyperlipemic serum to the incubation medium did not increase the incorporation fo either 3H-labelled oleic acid or of 14C-labelled linoleic acid into phospholipid or triglyceride. The removal of labelled lipid fractions has also been followed for four days in cells pulse labelled for 24 hours with 3H-labelled oleic acid and 14C-labelled linoleic acid. Both 3H- and 14C-labelled cholesterol esters were removed more rapidly when the smooth muscle cells were grown in medium containing normal serum than in medium containing hyperlipemic serum. The removal of both phospholipid and triglyceride was similar in normal and hyperlipemic serum. Comparison of the 3H/14C ratio indicated that the cholesterol oleate and cholesterol linoleate were removed at similar rates.     

Acetylpolyamines decrease blood pressure, [Ca++]i and isometric force of vascular smooth muscle

Polyamines are polycations in cells and acetylation is the first step in their intracellular metabolism. We investigated the effects of the acetylated polyamines on arterial blood pressure and vascular reactivities in rats. Acetylspermine and acetylspermidine, administered at concentrations ranging from 12.5 to 50 mmol/kg b.wt., both induced a transient decrease in mean arterial blood pressure. The magnitudes of the hypotensive effects of these acetylpolyamines and polyamines were in the order of spermine > acetylspermine > acetylspermidine = spermidine. Pretreatment of rats with calcium diminished polyamine-induced hypotensive effects. The effects of spermine and acetylspermine on isolated vascular smooth muscle were examined in rat aortic rings and tail artery strips. Both compounds relaxed precontracted arterial preparations, and this relaxation could be counteracted by increasing extracellular calcium concentration. Tail artery strips were more sensitive to acetylspermine when compared to aortic rings. In tail artery strips preloaded with the bioluminescent protein aequorin, both spermine and acetylspermine caused a concomitant decrease in intracellular calcium and isometric force activated by 36 mM of KCl. These results demonstrate clearly that acetylspermine and spermine alike decrease intracellular calcium concentration of vascular smooth muscle, which is likely to account for the relaxation of vasculature. The relaxation of smooth muscle in the vascular wall in turn might lead to decreased arterial blood pressure.     

Enhanced inhibition by melatonin of alpha-adrenoceptor-induced aortic contraction and inositol phosphate production in vascular smooth muscle cells from spontaneously hypertensive rats

PURPOSE: To theoretically derive and empirically validate the relationship between the actual thick intraocular lens and the thin lens equivalent. METHODS: Included in the study were 12 consecutive adult patients ranging in age from 54 to 84 years (mean +/- SD, 73.5 +/- 9.4 years) with best-corrected visual acuity better than 20/40 in each eye. Each patient had bilateral intraocular lens implants of the same style, placed in the same location (bag or sulcus) by the same surgeon. Preoperatively, axial length, keratometry, refraction, and vertex distance were measured. Postoperatively, keratometry, refraction, vertex distance, and the distance from the vertex of the cornea to the anterior vertex of the intraocular lens (AV(PC1)) were measured. Alternatively, the distance (AV(PC1)) was then back-calculated from the vergence formula used for intraocular lens power calculations. RESULTS: The average (+/-SD) of the absolute difference in the two methods was 0.23 +/- 0.18 mm, which would translate to approximately 0.46 diopters. There was no statistical difference between the measured and calculated values; the Pearson product-moment correlation coefficient from linear regression was 0.85 (r2 = .72, F = 56). The average intereye difference was -0.030 mm (SD, 0.141 mm; SEM, 0.043 mm) using the measurement method and +0.124 mm (SD, 0.412 mm; SEM, 0.124 mm) using the calculation method. CONCLUSION: The relationship between the actual thick intraocular lens and the thin lens equivalent has been determined theoretically and demonstrated empirically. This validation provides the manufacturer and surgeon additional confidence and utility for lens constants used in intraocular lens power calculations.     

Glucose-induced oxidative stress in vascular contractile cells: comparison of aortic smooth muscle cells and retinal pericytes

Once onset of clinical rabies develops in an individual, death is inevitable. Thus, it is imperative that, for persons exposed or potentially exposed to rabies virus, prophylaxis must be instituted as soon as possible following the exposure. Local wound management is an essential part of postexposure rabies prophylaxis. Exposed persons should receive a recommended series of a tissue culture or cell culture origin vaccine. The number of doses and route of vaccination differ in various regions of the world and are discussed in the text. The administration of a rabies immune globulin is generally recommended in conjunction with the first dose of the rabies vaccine. Nerve tissue origin vaccines, although used extensively in some parts of the world, are not recommended if cell or tissue culture vaccines are available. Decision trees are presented in the text to aid in determining if rabies vaccine is necessary following a known or presumed exposure to the virus, along with a table outlining the various rabies vaccines available in the World. Rabies pre-exposure immunisation is recommended for those individuals at risk of exposure to the virus. Pre-exposure prophylaxis consists of 3 doses of an approved rabies vaccine administered either intramuscularly or intradermally on days 0, 7, and 21 or 28 with periodic booster doses or titre determination depending on the level of risk of potential exposure to the virus.