Constance P. Christian

Ontogeny of Apoptosis during Lung Development

Apoptosis has been shown to be involved in several processes during embryogenesis, but the ontogeny of apoptosis during lung development has not been studied. The goals of the current study were to determine if apoptosis occurs during lung development, and to determine the ontogeny of the changes in apoptosis that occur. We studied the ontogeny of apoptosis in vivo using lungs from 14-18-d gestation fetal rats, newborn rats, and 1-d-, 2-d-, 5-d-, and 10-d-old rat pups. Apoptosis was assessed by electron microscopy and the terminal deoxyribonucleotidyl transferase dUTP nick end-labeling assay. We compared the in vivo results with explants of 14-d gestation fetal rat lung placed in culture for 1-4 d because the biochemical development of the lung in organ culture has been shown to closely parallel the development of the lung in vivo. We found apoptosis of mesenchymal cells at the periphery of distal lung buds in early fetal lung(14-16-d gestation). Apoptosis of both mesenchyme and epithelium was present in later fetal lung (18-d gestation). There were no qualitative differences in apoptosis between in vivo fetal lung and explant cultures of fetal lung. There was a 14-fold increase in apoptosis at birth and in the first postnatal day of life (9-12% of cells) compared with fetal lung (0.6-1% of cells). This was followed by a rapid decline in the percentage of apoptotic cells to fetal levels at postnatal d 2-10. We conclude that apoptosis occurs in a spatially, temporally, and cell-specific manner during lung development. The number of cells undergoing apoptosis increases dramatically in the first day after birth.

Cytochemical localization of adenylate cyclase.

This study examined 1) the effect of lead and fixation on adenylate cyclase activity, 2) the effect of lead on App(NH)p, and 3) the specificity of App(NH)p as a substrate for adenylate cyclase under the conditions of the cytochemical assay. The results indicated that: 1) fixation that provides adequate structural preservation inhibits enzyme activity to varying degrees depending on the tissue, fixative, length and temperature of fixation; 2) millimolar concentrations of lead do not negatively affect the adenylate cyclase activity of several different tissues (especially if 10 mM NaF is present); 3) lead does not cause the nonenzymatic hydrolysis of App(NH)p; 4) the App(NH)p obtained from the supplier is contaminated and should be purified before use, since lead can interact with the contaminants and this may be a source of error in the assay; and 5) adenylate cyclase appears to be the major enzyme that cleaves App(NH)p under cytochemical conditions.

Ultrastructural studies of the rat submandibular gland in streptozotocin induced diabetes mellitus

Increased fluid intake (polydipsia) is one of the classic symptoms of diabetes mellitus. Xerostomia (dry mouth) and resultant thirst are other symptoms of the disease and bear a close relationship to polydipsia. The xerostomia in individuals with diabetes is primarily due to decreased saliva flow which appears to be associated with degenerative changes in the salivary glands. This study examines the response of the rat submandibular gland to streptozotocin induced diabetes mellitus. Adult male rats were given a single I.V. dose of streptozotocin (65 mg/kg body weight) in citrate buffer (pH 4.5). Salivary glands were examined by light and electron microscopy at 4, 8 and 24 h and 3, 7, 14 and 21 days posttreatment. The changes in the acinar cells were characterized by an accumulation of secretory material within the cytoplasm. This secretory protein accumulation was followed by degenerative changes in the acinar cells which frequently resulted in cell death and replacement of secretory cells by connective tissue elements. The loss of secretory volume and potential changes in secretory kinetics are discussed with regard to the xerostomia, thirst and polydipsia exhibited by individuals with diabetes mellitus.

Chapter 32 Development of Stimulus—Secretion Coupling in Salivary Glands

Publisher Summary This chapter provides evidence that the secretory cells of the developing rat submandibular gland (SMG) acquire the ability to synthesize and package secretory proteins prior to attaining the ability to release the packaged product in response to hormonal stimuli. The chapter correlates the development of the secretory response with studies on hormonal activation of cell surface–associated adenylate cyclase and with direct measurements of β -adrenergic and α -adrenergic binding sites. The chapter thus provides a picture of the development of the stimulus–secretion coupling system in this model exocrine system. Electrophysiological and morphological evidence indicates that in this system, development of the stimulus–secretion-coupling mechanism precedes the establishment of the neural connections that regulate secretion in vivo . The data presented in the chapter give a comprehensive picture of the maturation of those factors that regulate protein secretion in this model exocrine system. It appears that differentiating SMG secretory cells first develop the structural and synthetic machinery required to produce and package their secretory product.

The influence of the sympathetic nervous system on the development of β-adrenergic receptors in the rat submandibular salivary gland

During the development of the rat submandibular gland (SMG) there is a clearly-defined sequence in the maturation of the beta-adrenergic receptor/adenylate cyclase-linked stimulus-secretion coupling system. The sympathetic nervous system does not become functionally linked to the exocrine process in the SMG until approximately six days after birth. The temporal correlation of the ingrowth of catecholamine-containing nerve processes, the appearance of beta-adrenergic receptors and the functional coupling of the stimulus-secretion system suggested the possibility of a cause and effect relationship between the appearance of the catecholamine-containing nerves in the gland and the maturational increase in the number of beta-adrenergic receptors. Chemical sympathectomy in neonates did not effect the time of appearance or the number of beta-adrenergic receptors seen in the developing gland. However, chronic isoproterenol treatment resulted in accelerated maturation of the gland with a concomitant premature appearance of the beta-receptors. These data suggest that the increase in the number of beta-adrenergic receptors which normally occurs in the developing gland at 5-6 days after birth is a specifically-programmed step closely associated with the degree of maturation attained by the cells and is independent from the ingrowth of catecholamine-containing nerves.

Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units.

The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.

Effects of reserpine treatment on β-adrenergic/adenylate cyclase modulated secretion and resynthesis by the rat submandibular gland

Chronic reserpine (adrenergic blocking) treatment causes a marked accumulation of secretory protein in the rat submandibular gland (SMG) but discharge of this material is delayed in response to isoproterenol stimulation. The purposes of this study were to investigate the effects of chronic reserpine treatment on 1) the number of β-adrenergic receptors, 2) the sensitivity of cell-surface-associated adenylate cyclase to various concentrations of isoproterenol, and 3) to correlate these data to morphologic studies of the secretion and resynthesis phases of the isoproterenol-induced secretory cycle in the rat SMG. Animals were injected with reserpine (0.5 μg/g b.w.) for 6 days. Plasma membrane fractions were prepared. The adenylate cyclase response to a series of isoproterenol concentrations, and the number of β-adrenergic receptors ([3H]-alprenolol binding) were determined. Other animals were given a single dose of isoproterenol (0.8 mg/100 g b.w.) and the SMG was examined by light and electron microscopy at various times (30 min to 24 h) after treatment. Chronic reserpine treatment leads to a 2.5-fold increase in SMG β-adrenergic binding sites and a 50-fold increase in adenylate cyclase sensitivity to IPR stimulation when compared to controls. However, secretion and resynthesis of secretory product in response to IPR stimulation was greatly delayed in reserpinized rats.

Inhibition of rat salivary gland adenylate cyclase by glycosaminoglycans and high molecular weight polyanions

The effects of hyaluronic acid, chondroitin-4 and -6 sulphate, dermatan sulphate, heparin and the sulphated, polysugar dextran sulphate on membrane-associated adenylate cyclase were investigated in a plasma-membrane fraction derived from the glands. Adenylate-cyclase activity was inhibited in a dose-dependent fashion by all. The potency (concentration causing a 50 per cent inhibition (Ki) of adenylate cyclase activity) of each molecule varied with the degree of sulphation of the agent tested. The GTP and Gpp(NH)p activation of adenylate cyclase as well as basal-enzyme activity and of adenylate cyclase by isoproterenol (a beta-adrenergic agonist) were inhibited; however, little effect on hormone binding (assessed by [3H]-dihydroalprenolol binding) was observed at Ki concentrations. Inhibition of NaF-activation of adenylate cyclase was not as pronounced as the other inhibitory effects. The findings suggested that the agents inhibited enzyme activity by action at or near the catalytic site; hence the effect of these molecules on forskolin activation of adenylate cyclase was investigated. The polyanions inhibited forskolin stimulation of the enzyme to the same degree that they had previously inhibited basal, GTP and hormonally-stimulated enzyme activity. Thus, these molecules inhibited adenylate cyclase by an action (perhaps charge-related) that directly effected the stabilization of catalytic site of the enzyme. Thus polyanions of the type tested can directly modulate adenylate-cyclase activity and may serve as potential regulatory molecules for various biologic functions which are cyclic-AMP dependent.

Effects of salmeterol on secretion of phosphatidylcholine by alveolar type II cells.

Beta-adrenergic agents enhance secretion of phosphatidylcholine (PC) by adult and fetal type II cells. We have previously shown that terbutaline stimulates secretion of PC by fetal type II cells, but the response wanes after 30 minutes. We studied the effects of salmeterol, a highly selective, long-acting beta2-adrenergic agonist that does not cause receptor desensitization, on PC secretion by adult type II alveolar cells in primary culture. Release of lactate-dehydrogenase was < 4% and did not vary with the concentration of salmeterol. Salmeterol stimulated PC secretion in a concentration-dependent manner. The maximum effective-concentration tested was 50 nM and the EC50 was 11.40 +/- 1.14 nM. Propranolol inhibited the effect of salmeterol on release of PC, confirming that the effects of salmeterol are mediated by beta-receptors. OT50, the time for onset of action, was 32.0 +/- 1.6 minutes. RT50, the time to achieve 50% recovery from maximal stimulation was, 393.0 +/- 20.2 minutes. We conclude that salmeterol stimulates PC secretion by type II cells through activation of beta-adrenergic receptors and has a longer duration of action (>6 hours) compared to other beta2-agonists. Salmeterol may be a useful drug with which to study the role of receptor desensitization in the developmental changes in PC secretion.

Animal Models of Sjögren's Syndrome

Sjigrens Syndrome (SS) is an autoimmune disease which occurs predominately in women over 40 years of age. Typically, patients present with complaints of dry mouth and eyes which often lead to painful stomatitis, an increased incidence of caries and periodontal disease, difficulties in speech and swallowing, as well as the more prominent keratoconjunctivitis sicca (see Moutsopoulos, 1980, for review). SS is associated with the major histocompatibility complex (HLA), and patients have a high incidence of other autoimmune disorders. The disease is characterized by infiltration of the salivary and lacrimal glands by T and B lymphocytes, and four separate auto-antibodies have been found in the sera of Sjbgrens patients. An appropriate animal model of SS [Experimental Autoallergic Sialadenitis (EAS)J is needed to identify the target antigens and define the immune mechanisms which arc involved in the disease. However, few animal models of SS arc available. There is a limited number of spontaneous murine models (Hoffman et of., 1984) and an inducible rodent model (White and Casarett. 1974). In the present study, we have evaluated some of the available animal models of SS.