Gary L. Huber

Synthesis of Dipalmitoyl Lecithin by Alveolar Macrophages

A reliable, relatively simple method for isolation and quantification of disaturated lecithins is described. In rabbit lung, 34% of the lecithins were disaturated, in alveolar macrophages, 19%. More than 95% of the fatty acids of the disaturated lecithins from lung and alveolar macrophages was palmitic. Hence, the disaturated lecithins from these sources were essentially all dipalmitoyl lecithin. Both heterophils and alveolar macrophages incorporated (14)C-labeled choline and palmitate into disaturated lecithins. Liver slices in which only about 1% of the lecithins were disaturated incorporated very little of these precursors into this fraction. Of the palmitate incorporated in vitro into disaturated lecithins by alveolar macrophages, heterophils, and lung slices, 37% was in the 1 position. In disaturated lecithins isolated from pulmonary lavage fluid, alveolar macrophages, and lung of rabbit 8-12 hr after a single intravenous injection of palmitic-1-(14)C acid, 45% of the (14)C was in position 1. At earlier times, from 20-240 min after injection, the distribution of (14)C was similar in the samples from lung, but in those from alveolar macrophages and lavage fluid, the percentage in position 1 was slightly lower.Glycerol-U-(14)C was incorporated into disaturated lecithins by alveolar macrophages and by lung slices in vitro. Both tissues incorporated very little label from ethanolamine or from methyl-labeled methionine into this fraction. All of the data are consistent with the view that alveolar macrophages synthesize dipalmitoyl lecithin via the cytidine diphosphate-choline pathway.

Quantitative differences in goblet cells in the tracheal epithelium of male and female rats.

The cause of higher morbidity rates among men than women from diseases of the respiratory tract may be complex and involve several factors, one of which may be structural differences within airways. In an attempt to determine whether such differences exist, 13 male rats and 45 female rats (15 each in proestrous, estrous, and diestrous) were evaluated. Tracheas were fixed in situ by injection of a mixture of formaldehyde and glutaraldehyde. Longitudinal sections cut from paraffin blocks were stained and combined alcian blue (pH 2.5) and periodic acid-Schiff stain to count goblet cells, or with Weigerts hematoxylin and eosin stain to determine epithelial thickness. In the normal rat trachea, cells containing periodic acid-Schiff-positive granules at their apices constituted a major population of goblet cells. The number of these goblet cells present in the trachea was greater in female than in male rats at each stage of the estrous cycle, with the values in the diestrous females closest to the value in the males. Among females, estrous and proestrous rats contained significantly more of these goblet cells than did diestrous animals. The tracheal epithelium of male rats was significantly thicker than that of female rats. These studies demonstrate that the tracheal epithelium differs in male and female rats and varies in the female rat as a function of hormonal cycles. These results raise the question of whether similar differences might be important in the pathogenesis of human disease, and they deserve further clarification in man.

Contractile performance of rat myocardium after chronic tobacco smoke inhalation.

The mechanical performance of isolated left ventricular muscle preparations from rats exposed to smoke from Kentucky Reference cigarettes was examined for possible chronic effects. Eight rats were subjected to smoke for periods of 10 min/hr for 5 hr/day for 180 days. Nineteen additional rats served as either sham-smoked controls or weight-matched, food-deprived controls. Rats exposed to tobacco smoke had a significant diminution in body and left ventricular weight compared to sham-smoked controls. When compared to food-deprived rats, no differences in weights were observed. Contraction mechanics were measured for each muscle at the peak of its length tension curve. No significant difference in cardiac muscle performance was found in rats exposed to tobacco smoke when compared to control animals with respect to contractile performance under oxygenated conditions, muscle performance during 60 min of hypoxia or subsequent reoxygenation, or sensitivity of mechanical performance to isoproterenol. Thus, chronic cigarette smoke exposure did not alter the intrinsic mechanical performance of isolated rat ventricular muscle.

Stereology of lavaged populations of alveolar macrophages: effects of in vivo exposure to tobacco smoke.

Abstract A multi-level morphometric and stereologic study was performed on alveolar macrophages obtained by bronchopulmonary lavage of unexposed control rats and rats experimentally exposed to whole tobacco cigarette smoke for 90 consecutive days. Measurements of macrophage profile diameters made at the light microscope were used to derive cell size distribution curves, with a mean cell volume in the control group of 623 μm3. At the electron microscope level, the volume density and numerical density of mitochondria in unexposed control cells were lower than the values reported for peritoneal macrophages, but the mean volume of individual mitochondria was five times higher in the alveolar than in the peritoneal macrophage. The volume density of lysosomes was slightly higher in alveolar than in peritoneal macrophages. Exposure to smoke resulted in changes in several subcellular compartments, including a 2-fold increase in mean cell volume, a 19-fold increase in the volume density of lipid inclusions, an increase in the surface density of mitochondrial inner membrane and a decrease in the surface density of rough endoplasmic reticulum. The significance of these morphologic changes in terms of cell function is discussed.

Tobacco smoke. Effects on pulmonary host defense.

Tobacco smoke affected both the metabolism and function of pulmonary alveolar macrophages (PAM). Phagocytosis of viable Staphylococcus aureus and inert starch particles was minimally but consistently depressed in PAM from rats exposed to tobacco smoke for six months. Oxygen consumption, superoxide and hydrogen peroxide release, and hexose monophosphate shunt activity were elevated in cells from smokers. Oxidation of glucose, labelled in the carbon-six position, remained unchanged. All observed effects of tobacco smoke on oxygen metabolism occurred during phagocytosis and did not affect the basal metabolism of the nonstimulated cell.Tobacco smoke affected both the metabolism and function of pulmonary alveolar macrophages (PAM). Phagocytosis of viableStaphylococcus aureus and inert starch particles was minimally but consistently depressed in PAM from rats exposed to tobacco smoke for six months. Oxygen consumption, superoxide and hydrogen peroxide release, and hexose monophosphate shunt activity were elevated in cells from smokers. Oxidation of glucose, labelled in the carbon-six position, remained unchanged. All observed effects of tobacco smoke on oxygen metabolism occurred during phagocytosis and did not affect the basal metabolism of the nonstimulated cell.

An experimental animal model for quantifying the biologic effects of marijuana on the defense system of the lung.

An animal model was developed as an inhalation bioassay for acute and chronic delivery of tobacco and marijuana smoke in physiologic doses to the lungs of experimental animals. Although generating identical aerosols, the pyrolyzation characteristics of marijuana and tobacco differed considerably. Both products impaired host intrapulmonary antibacterial defenses, with marijuana appearing in comparable dosages to be significantly more toxic to the alveolar bactericidal activity of the lung than tobacco smoke. The effect of both smoking products on mucociliary clearance by the airways, in this model system, appeared to be negligible. Direct biologic comparisons between the effects of the two smoking products must be guarded, however, as dosimetry cannot be precisely equated because of their opposing physiologic effects on the lung.

Successful Smoking Cessation

Tobacco use will kill as many as 1 billion people in the 21st century unless its use is curtailed. Smoking cessation is associated with several health and personal benefits; however, smoking cessation is difficult because of the highly addictive nature of nicotine.Successful smoking cessation strategies can generally be divided into two groups: nicotine replacement therapy (NRT) and behavioral modification. The National Cancer Institute in the US advocates the use of the ‘Ask-Assess-Advise-Assist-Arrange’ approach to strengthen the role of the healthcare provider in initiating and sustaining smoking cessation. The ‘stages of change’ model is also useful in tailoring the physician’s approach to smoking cessation to the patient’s attitude to this change.There are several approved types of NRT. These include nicotine polacrilex gum, transdermal nicotine patches, nicotine lozenges, nicotine nasal sprays, nicotine inhalers, and nicotine lollipops. Nicotine polacrilex gum, nicotine transdermal patches, and nicotine lozenges are available both on prescription and over the counter. Nicotine nasal sprays and nicotine inhalers are available by prescription only, while nicotine lollipops are compounded by pharmacists. One of the drawbacks of these therapies is that they can be used in excess of recommended doses or durations of therapy. They are also all subject to the adverse effects of nicotine, which include dizziness, altered cardiac rhythms, nausea and vomiting, fatigue and muscle aches, and headache. Orally administered NRT is also associated with the additional adverse effects of sore throat, mouth sores, esophageal irritation, and stomach pain, while nicotine inhalers are associated with throat and airway irritation and coughing; nicotine nasal sprays are associated with a runny nose, sneezing, throat irritation, and coughing early in the course of treatment; and transdermal nicotine patches are associated with skin irritation.More recently, other pharmacological treatments have been used for smoking cessation. Bupropion was developed as an antidepressant, but has been shown to reduce the desire to smoke. Bupropion may be used in combination with NRT and may also be helpful in reducing post-cessation weight gain. Varenicline is a specifically designed smoking cessation medication. It has been shown to greatly reduce both smoking withdrawal symptoms and the desire to smoke. However, varenicline has been associated with mood alteration that may be as severe as to include suicidality, gastrointestinal adverse effects, and sleep disturbances. Other pharmacological and non-pharmacological therapies have been used in smoking cessation, with variable effects.Smoking cessation is difficult and ‘staying quit’ is even more so. Most individuals ‘quit’ more than once before they become tobacco free. Two major factors in this are the addictive nature of nicotine and post-cessation weight gain. Successful smoking cessation may require combinations of pharmacological therapies and behavioral modification. Therapies may need to be used in excess of recommendations. Tobacco smoking is a lethal addiction. No effort should be spared in achieving successful smoking cessation.

Acute blood loss and pulmonary host defense mechanisms in the rat

Abstract After trauma and hemorrhage, there is a high incidence of bacterial pneumonia. We have evaluated in the rat the effect of acute blood loss on intrapulmonary host defense mechanisms. Rats were phlebotomized 2.0 ml/100 g body weight and were then kept without food or water until sacrifice 15 and 38 hr later. Intrapulmonary inactivation of an inhaled inoculum of radiolabeled ( 32 P) S. aureus was quantified in individual rats 14 hr after exposure in an aerosol generator system. Since dehydration and starvation by themselves might influence pulmonary phagocytic activity, separate experiments were included to evaluate these factors. The acute blood loss produced hypotension for less than 1 hr. When sham and experimental rats were challenged with S. aureus 1 hr after surgery, the phlebotomized rats had an impaired intrapulmonary bacterial inactivation when sacrificed 14 hr later. This was unrelated to changes in lung water and arterial pH, pCO 2 and pO 2 . Sham and experimental rats exposed to S. aureus 24 hr after surgery had a similar depression of intrapulmonary bacterial inactivation which was related to decreases in lung water. Starvation had no effect on intrapulmonary bacterial inactivation but 38 hr of dehydration caused an impairment which was related to decreased lung water. After acute blood loss, there is a direct effect of hypotension on intrapulmonary host defense mechanisms. Since blood loss did not cause a greater impairment of bacterial inactivation at 38 hr than dehydration alone, the factor operational in the earlier hours after hemorrhage may have no longer been present. Both dehydration and blood loss may be operative in the traumatized patient and contribute to the development of posttraumatic pulmonary infections.

Tobacco smoke and the pulmonary alveolar macrophage.

Our results indicate that tobacco smoke exposure to varying duration causes morphological, biochemical and functional alterations in pulmonary alveolar macrophages. The results of these changes is a population of alveolar macrophages made up of larger cells, with a reduced nucleus-cytoplasmic ratio, which are heavily loaded with heterolysosomes containing lipid. Though their fractional complement of mitochondria remains the same, an increase in the inner mitochondrial membrane surface area may be related to an enhanced oxidative metabolism. The cell is biochemically activated particularly following chronic exposure and is functionally impaired with respect to phagocytosis.

The direct effect of tobacco smoke on the intrinsic mechanical properties of cardiac muscle

Abstract The direct effects of tobacco smoke from Kentucky University Reference cigarettes on the mechanical performance of isolated rat left ventricular muscle preparations were examined. Experiments were carried out with preparations contracting isometrically 12 times per minute at 28°C while at the apex of the length tension curve. Under oxygenated conditions, tobacco smoke bubbled through the bath induced an initial negative inotropic response. With larger volumes of smoke, initial depression was followed by recovery toward control values. Late recovery was inhibited by first passing the tobacco smoke through a Cambridge filter. Tobacco smoke added prior to 60 min of hypoxia, followed by 30 min of reoxygenation, had a dose-dependent deleterious effect on muscle performance during hypoxia and subsequent reoxygenation. The deleterious effect of smoke during hypoxia could largely be prevented by first passing the tobacco smoke through a Cambridge filter. The nicotine present in tobacco smoke could not by itself account for the changes under oxygenated conditions or those seen during hypoxia and reoxygenation. It is concluded that the gaseous phase of tobacco smoke is responsible for the negative inotropic effect seen under oxygenated conditions. The action of the particulate phase of the smoke (which is blocked at least in part by the Cambridge filter) appears to be responsible for the partial recovery of mechanical performance under oxygenated conditions and the potent deleterious effects seen during hypoxia and reoxygenation.