Loss of SM-B myosin affects muscle shortening velocity and maximal force development.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation.

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [14C]sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t1/2 = 5 min in macrophages, 6--8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t1/2 = 180 min in macrophages, 430--620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercompartment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min.

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids.

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts.

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.

Mammalian ribosomal protein: analysis by electrophoresis on polyacrylamide gel.

Ribosomal protein from five mammalian tissues when analyzed by discontinuous electrophoresis on polyacrylamide gel at pH 4.5 yielded 24 bands. Densitometric tracings indicated that the patterns of the basic ribosomal proteins from the several tissues were qualitatively similar. Protein from Escherichia coli ribosomes analyzed at pH 4.5 gave 29 bands, and the pattern was different from that of mammalian ribosomal protein. No distinct band was found when mammalian ribosomal protein was analyzed at pH 8.3 (acidic proteins). Ribosomal protein from Escherichia coli gave eight bands at pH 8.3. Thus, the structure of the genes responsible for synthesis of ribosomal protein in several mammalian tissues is the same, and different genes direct synthesis of ribosomal protein in bacteria.

Protein synthesis in pulmonary alveolar macrophages. Source of amino acids for leucyl-tRNA.

Extracellular, intracellular and tRNA-bound leucine pools of the adherent pulmonary alveolar macrophage were examined to determine the relationships between them and the precursor for protein synthesis. When cells were cultured in media of various leucine concentrations, the patterns of isotope distribution in intracellular and extracellular leucine did not correlate with the patterns seen in protein-bound leucine. hence, the free leucine pools cannot be used reliably as precursors for calculating rates of protein synthesis. tRNA-bound leucine, however, behaved isotopically as if it were the precursor. Constant synthetic rates were calculated using the tRNA specific activity over a wide range of leucine concentrations. In addition, by measuring the tRNA-bound specific activities of three different amino acids, leucine, valine and phenylalanine, and their respective specific activities in protein, we were able to calculate independently three separate but identical synthetic rates. At physiological amino acid concentrations, the macrophage intracellular leucine pool and the tRNA-bound leucine pool received less than half of their amino acids from extracellular sources. At 5 mM external leucine, the intracellular specific activity was indistinguishable from that of the medium leucine, but the specific activity of the tRNA-bound leucine pool remained only about 50% that of the extracellular value. The most straightforward interpretation of why the tRNA-bound leucine did not flood with external label under conditions where the intracellular pool has reached equilibrium is to propose that some portion of the leucine for protein synthesis is derived directly from protein turnover before the degradation products have mixed with the common amino acid pool.

Increased collagen synthesis and decreased collagen degradation in right ventricular hypertrophy induced by pressure overload.

OBJECTIVE: The aim was to examine the effect of pressure overload in rabbits on ventricular collagen metabolism and procollagen gene expression. METHODS: Right ventricular hypertrophy was induced by banding the pulmonary artery such that the diameter of the vessel was reduced by 50%, and animals killed in groups after two and 14 days. Collagen synthesis and degradation of newly synthesised collagen were assessed following a single intravenous injection of 3H-proline with a flooding dose of non-radioactive proline, given 3 h before the animals were killed. Northern and slot blot analyses were performed to measure procollagen alpha 1(I) mRNA. RESULTS: The fractional collagen synthesis rate increased sixfold in the right ventricle only 2 d after pulmonary artery banding (p < 0.001), then fell to just over double the control value by 14 d (p < 0.05 from control). The proportion of newly synthesised collagen degraded decreased from 50.7(SD 12.8)% to 26.8(15.8)% in 2 d (p < 0.05) and remained at this level. The procollagen alpha 1(I) mRNA level increased by more than fourfold in the right ventricle 2 d after the onset of pressure overload, and was less than three times control levels at 14 d. CONCLUSIONS: The development of right ventricular hypertrophy is associated with a rapid increase in collagen production, with regulation at multiple sites in the biosynthetic pathway. This regulation occurs at both transcriptional and post translational levels.

Lung smooth muscle differentiation.

The vascular and visceral smooth muscle tissues of the lung perform a number of tasks that are critical to pulmonary function. Smooth muscle function often is compromised as a result of lung disease. Though a great deal is known about regulation of smooth muscle cell replication and cell and tissue contractility, much less is understood regarding the phenotype of the contractile protein machinery of lung smooth muscle cells. This review focuses on the expression of cytoskeletal and contractile proteins of lung vascular and airway smooth muscle cells during development, in the adult and during vascular and airway remodeling. Emphasis is placed on the expression of the heavy chain of smooth muscle myosin, as well as the regulation of its gene. Important areas for future research are discussed.

Lectin binding patterns to terminal sugars of rat lung alveolar epithelial cells.

We used post-embedding cytochemical techniques to investigate the lectin binding profiles of rat lung alveolar epithelial cells. Sections from rat lung embedded in the hydrophilic resin Lowicryl K4M were incubated either directly with a lectin-gold complex or with an unlabeled lectin followed by a specific glycoprotein-gold complex. The binding patterns of the five lectins used could be divided into three categories according to their reactivity with alveolar epithelial cells: (a) the Limax flavus lectin and Ricinus communis I lectin bound to both type I and type II cell plasma membranes; (b) the Helix pomatia lectin and Sambucus nigra L. lectin bound to type II but not type I cells; and (c) the Erythrina cristagalli lectin reacted with type I cells but was unreactive with type II cells. The specificity of staining was assessed by control experiments, including pre-absorption of the lectins with various oligosaccharides and enzymatic pre-treatment of sections with highly purified glycosidases to remove specific sugar residues. The results demonstrate that these lectins can be used to distinguish between type I and type II cells and would therefore be useful probes for investigating cell dynamics during lung development and remodeling.

Glucagon and phenylephrine combination vs glucagon alone in experimental verapamil overdose.

OBJECTIVE: To evaluate glucagon and phenylephrine in combination as a treatment for the hemodynamic effects of verapamil overdose. METHODS: Pentobarbital-anesthetized and instrumented dogs were overdosed using a previously developed verapamil overdose model (15 mg/kg IV over 30 minutes). The animals were maintained and observed for 90 minutes or until death. Cardiac output (CO), heart rate (HR), and mean arterial pressure (MAP) were monitored. Following the 30-minute verapamil infusion (toxicity), the control animals received no treatment; the glucagon animals received a 5-mg glucagon bolus and a drip of 5 mg/90 minutes; and the glucagon/phenylephrine animals received the same glucagon therapy plus a phenylephrine drip titrated to 180 micrograms/min over 15 minutes. The groups were compared using analysis of variance: the experimental variables were group and time; the response variables were changes from toxicity for the hemodynamic parameters. Post-hoc comparisons were done with alpha set at 0.05. RESULTS: A significant change in CO was seen in the glucagon group (delta = 2.6 L/min) and the glucagon/phenylephrine group (delta = 1.9 L/min) compared with the control group (delta = 0.8 L/min). The change in CO was significantly larger for the glucagon animals compared with the glucagon/phenylephrine animals. The change in MAP for the glucagon/phenylephrine group (delta = 24 mm Hg) was significant compared with the control group (delta = 14 mm Hg). The MAP change for the glucagon group (delta = 19 mm Hg) was not significantly different from that of either the control or the glucagon/phenylephrine group. The change in glucagon HR (delta = 6 beats/min) was significant compared with the control group (delta = -4 beats/min) and the glucagon/phenylephrine group (delta = -4 beats/min). CONCLUSION: The glucagon/phenylephrine therapy improved MAP compared with the control, but reduced CO and HR compared with glucagon alone. Glucagon/phenylephrine therapy is not as effective as glucagon alone in reversing the hemodynamic effects of experimental verapamil overdose.

The effect of d-amphetamine and ephedrine on smoking attitude and behavior.

Using a double blind, randomized, latin square design, 17 light smokers and 6 heavy smokers were given three times per day doses of placebo, 5 mg amphetamine sulfate, 7.5 mg amphetamine sulfate, 25 mg ephedrine hydrochloride or 50 mg ephedrine hydrochloride. Compared to placebo, active drug produced a statistically significant drop in feeling of addiction to cigarettes (p = 0.022). Ephedrine was reported to be more effective than amphetamine (p = 0.046). Subjects reported similar changes in feeling of enjoyment of smoking. Active drug produced a statistically significant drop in the actual amount of tobacco smoked in heavy smokers (p = 0.028), but not in light smokers. Only two smokers were able to quit completely during the experiment, and one of those people resumed smoking after she stopped taking medication. Possible explanations of these findings are discussed.

Emergency air transport of pregnant patients: the national experience.

There is considerable debate as to the appropriate role of helicopter transfer of sick patients. The debate over helicopter transfer of high-risk obstetric patients is even more intense. There is clear evidence that high-risk neonates are more likely to survive when they are delivered in a perinatal center compared with local delivery followed by transfer. On the other hand, there is concern that in-flight delivery entails "extreme risks, both to mother and child." In spite of this debate, there has been little research into the frequency of delivery during transport to a hospital or into the mortality associated with such a delivery. A study involving a single center suggests that the incidence of in-flight delivery is very low. We contacted all American Society of Hospital-Based Emergency Air Medical Services (ASHBEAMS) member air ambulance programs to determine the national statistics for in-flight delivery and associated perinatal mortality. We found no instances of in-flight delivery in 357 helicopter transports; 315 of these women were in active labor at the time of transport and 72 were in the accelerated phase of labor. There is evidence that these flights were screened for safety. Airplane flights generally took more time than helicopter flights (P less than .05), and there was one in-flight delivery during 88 airplane transfers. Also presented are additional data that suggest in utero transport of high-risk fetuses to a perinatal center by helicopter is cost effective.

Cigarette smoke components and alveolar macrophage protein synthesis.

A comparison was made of the effects of acrolein and aqueous cigarette smoke extracts on amino acid incorporation into protein by rabbit pulmonary alveolar macrophages. Studies were on cells maintained in vitro as adherent monolayers. Freshly prepared acrolein inhibited amino acid incorporation by significant amounts after approximately 30 min and aqueous smoke extracts after approximately 15 min of incubation. Fifty percent inhibition by acrolein occurred with a dose of 5.5 microgram acrolein/ml, an amount four times that in the amount of aqueous smoke extract required for 50% inhibition according to previously reported findings. Analysis by a dual-isotope technique and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the inhibitory effect of acrolein to be nonspecific, as had previously been found for aqueous smoke extracts. The presence of the sulfhydryl reagent cysteine, reduced the inhibitory effect of acrolein by 57.5%, but reduced inhibition induced by aqueous smoke extracts by only 12.2%. These results suggest the effects of acrolein are both quantitatively and qualitatively different than those of aqueous smoke extracts.

Effects of kaolinite on amino acid transport and incorporation into protein by rabbit pulmonary alveolar macrophages.

A study was made of the effects of kaolinite, an aluminum silicate found in cigarette smoke and in alveolar macrophages of cigarette smokers, on the in vitro function of rabbit alveolar macrophages. Macrophages lavaged by standard procedures were incubated as adherent monolayers in the presence or absence of kaolinite, and amino acid incorporation into protein and transport subsequently measured. In the presence of dialyzed serum, kaolinite slightly inhibited incorporation into protein during the first 2 to 3 hr of incubation, after which incorporation ceased and a large percentage of newly synthesized protein was released (50% effect at approximately 0.5 mg/ml kaolinite). A dual-isotope experiment indicated that any change in the synthesis of protein which may have occurred was not selective for any protein or group of proteins. Kaolinite also stimulated noncompetitive amino acid accumulation after 2 to 3 hr in the presence of serum. The effects of kaolinite were immediate when incubations were conducted in the absence of serum. Control experiments showed all of the effects of the aluminum silicate to be on the cells and not on the incubation medium. These results suggest that kaolinite is cytotoxic and exerts its effects by a mechanism similar to that proposed for magnesium silicates and silica, in which the naked silicate is immediately cytotoxic, but if coated with serum protein must first be uncoated by lysozomal enzymes before destroying the cells.