Intracellular calcium localization in stimulated and non-stimulated extraorbital lacrimal glands of rats

Acinar cells of extraorbital lacrimal glands from control, pilocarpinetreated, atropine-treated and atropine + pilocarpine-treated rats were studied using a potassium pyroantimonate technique and X-ray microanalysis for calcium localization at the ultrastructural level. This was done in order to identify intracellular compartmentalization of calcium and to elucidate any calcium translocation that might occur during the secretory process. Calcium-pyroantimonate complexes were identified in the mitochondria, plasma membrane and cytoplasmic vesicles of the untreated specimens and in the plasma membrane of atropine-treated specimens, these complexes decreased drastically in the actively-secreting cells. The function of calcium in lacrimal gland secretion and the action of pilocarpine and atropine on membrane calcium are discussed.     

Calcium signaling in lacrimal glands

Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca²⁺-mobilizing messenger, IP₃, and release of Ca²⁺ stored in the endoplasmic reticulum. The loss of Ca²⁺ from the endoplasmic reticulum then triggers a process known as store-operated Ca²⁺ entry, involving a Ca²⁺ sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro.     

Postnatal developmental changes in submandibular glands of rats and mice

The submandibular glands of mice and rats are not fully developed at birth. In early postnatal life, differentiation of acini takes place before that of granular convoluted tubule (GCT) cells. The latter develop from striated duct cells, and first appear in both species around 15 days of age. In mice their full development gets under way by 20 days of age and is rapid in males and slow in females, resulting in a clear sexual dimorphism in adults. In rats, GCT development is more protracted, and accelerates around 40 days of age, with no sexual dimorphism seen at any time. The course of postnatal development of several GCT cell products is correlated with the cytodifferentiation of these cells. Reliable data are available for the development of amylase, proteases (including kallikrein), renin, epidermal growth factor, and nerve growth factor. Preliminary information exists for a glucagon-like substance. Cytodifferentiation of GCT cells is under hormonal control. Androgens alone can not precociously induce GCT cells, but thyroid hormones can do so, acting either alone or synergistically with androgens.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

Structuro-functional units of the salivary and lacrimal glands

By means of the multilayer graphic and plastic reconstruction methods using series of semithin sections, spatial tridimensional organization of the epithelial complexes and blood microcirculatory bed in the rat palatal salivary glands and the lacrimal gland of the human newborn have been studied. Since their ducts serve not only for discharging their secrete into the external medium, but also for accumulation (as collectors), the sublobular unit--adenomere should be referred to as a part of elementary level of organization of the epithelial complexes. The adenomere has in its composition a collecting centrally situating duct. However, while studying structure of the blood microcirculatory bed, it is found out that there is not any strict territorial correspondence between its functional units and structural units of the glandular epithelium. Nevertheless, giving a great importance to a tight syntopic connection of the collecting ducts of the adenomeres with the postcapillary venules (that belong to filtrating microvessels), these are sublobular units--adenomeres that are distinguished as structural-functional units in the glands.     

Ultrastructural changes of sebaceous glands in castrated and testosterone-treated male rats

Summary The effect of testosterone on the sebaceous gland was studied in the male rat. Biopsies of dorsal skin from intact rats, from rats four weeks after castration, and from castrated rats treated with testosterone propionate for three weeks at a dose of 250 g/kg/day (s.c.) were examined by electron microscopy. In the treated animals intermediate sacrifices were performed on days 4,7,14,21. Stereology was used for a morphometric analysis of the smooth endoplasmic reticulum (SER). The presence of a vesicular endoplasmic reticulum throughout the cytoplasm of differentiating cells was observed in the sebaceous glands of intact rats. Following castration there was a shrinkage of these cells and a striking decrease in the volume density of the endoplasmic reticulum vesicles. The administration of testosterone to gonadectomized rats resulted in an increase in vesicle content above the normal level from the first week as revealed by stereological analysis. This study confirms the trophic effect of the androgen on the sebaceous gland at a subcellular level. Furthermore, it is shown that stereology is a useful method for detecting early hormone-induced changes and could be valuable for studying the effects of anti-androgens on this gland.     

Participation of the lacrimal glands in wound healing processes

A hypothesis is advanced that nociceptive stimulation activates the functional system of wound healing. Experiments were made to investigate whether the lacrimal glands, which are activated due to pain, are involved in the system of healing. Rat experiments have demonstrated that nociceptive stimulation of the lacrimal glands accelerates the repair of cutaneous wounds by 26 to 30%. Lacrimectomy accompanied by distension of wound rims (up to 26% by the 3d-4th hour) inhibits the rate of healing by 18 to 20%. Intraperitoneal injection of a lacrimal gland extract activates healing in lacrimectomized rats. The data obtained suggest that the lacrimal glands are involved in wound healing.     

Role of gap junctions in fluid secretion of lacrimal glands

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.     

Physiological changes in laboratory rats caused by different housing

Males rats of the Wistar strain (Institute of Physiology, CSAV ) were divided into six groups from the 30th to the 200th day of age. Rats (n = 54) were put into cages in various numbers (1, 2, 3, 4, 6 and 8 animals in a cage) and were weighed regularly and the consumption of water and food was measured. At the end of the experiment the animals were killed and the weight of some organs and haematological values were determined. During the experiment the highest weight was attained in animals with 3 or 4 members in one cage. The differences in weight were significant from the 80th day of age. The consumption of diets and water was not influenced significantly by the number of animals in a cage. On the 200th day, the differences between the groups were found in the weight of some organs, and in some haematological values.     

The effect of negative air ionization on the thymus glands of laboratory rats

Continuous negative air ionization, over two generations, was found to result in depletion of thymus mass when compared with control values. Evidence from adrenal weights suggested that this effect was not mediated via stress effect; it is suggested that there may be a direct action of negative ions on the immune system. The normal pattern of thymic growth and involution was unaffected by such treatment. However, successive generations of animals, raised in a negatively ionized atmosphere, showed no such depletion of thymus mass.     

Degenerative changes in the digestive glands of rats bearing Leydig cell tumors

We undertook this study to determine the effects of a transplantable Leydig cell tumor on the major digestive glands of the host rats. It has been known that after bearing this tumor for only two weeks, the rats become anorexic and cathectic, develop hypercalcemia and osteolysis, and their peripheral bone marrow becomes hyperplastic. We now demonstrate that the parotid glands undergo marked degeneration including depletion of secretory product, apparent loss of acinar organization and the appearance of conjoined nuclei. The submandibular and sublingual glands and the pancreas are virtually unaffected. The liver undergoes fatty degeneration, the Kupffer cells become more prominent and more numerous, and the sinusoids sometimes contain small islets of hemopoietic tissue.     

Characterization of functional melanotropin receptors in lacrimal glands of the rat

The specific melanotropin (MSH) binding sites of rat lacrimal glands were characterized with respect to anatomic distribution, peptide specificity and selectivity, and coupling to a biological response. Tissue distribution of MSH binding sites was determined by autoradiography following in situ binding of a radiolabeled, biologically active preparation of a superpotent alpha-MSH analog, [125I]-[Nle4,D-Phe7]-alpha-MSH ([125I]-NDP-MSH). Intense, specific (i.e., alpha-MSH-displaceable) [125I]-NDP-MSH binding was observed throughout lacrimal acinar tissue, but not in ducts or stroma. In freshly isolated lacrimal acinar cells, specific binding of [125I]-NDP-MSH was maximal within 30 min and rapidly reversible, with a dissociation half-time of about 15 min. A number of melanotropins [alpha-MSH, [N,O-diacetyl-Ser1]-alpha-MSH, [des-acetyl-Ser1]-alpha-MSH, beta-MSH, ACTH(1-24) and ACTH(1-39)] were recognized by these binding sites, as assessed by their inhibition of [125I]-NDP-MSH binding; NDP-MSH was the most potent (IC50 = 1.3 x 10(-9) M). In contrast, other peptides, including ACTH(4-10) and the nonmelanotropic peptides VIP, substance P, somatostatin, and ACTH(18-39) (CLIP), had no effects on tracer binding. In isolated lacrimal acinar cells, alpha-MSH and NDP-MSH stimulated intracellular cyclic AMP accumulation. We conclude that lacrimal acinar cells express functional receptors recognizing melanotropins, suggesting that the lacrimal gland may be a target for physiological regulation by endogenous melanotropins.     

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6)but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.