Dwight W. Warren

Androgen support of lacrimal gland function

The effects of dihydrotestosterone (DHT) (1 mg/kg) on biochemical parameters related to lacrimal secretion, basal tear flow rate, and pilocarpine-stimulated lacrimal gland fluid secretion, in mature ovariectomized rabbits were studied. The effects of the synthetic estrogen, diethylstilbestrol (DES) (100 μg/kg), on lacrimal gland biochemical parameters in normal mature female rabbits was also studied. Ovariectomy decreased the total serum levels of testosterone (T) by 88.5% and androstenedione by 35.9%, without changing the levels of dehydroepiandrosterone (DHEA) or its sulfate. Ovariectomy caused a significant regression of the lacrimal glands, decreasing total DNA by 35%, and total protein by 22%. DHT treatment of ovariectomized animals prevented lacrimal gland regression, increasing total gland DNA (31%) and total protein (18%). DHT treatment also increases Na+,K+-ATPase activity (29%) and ß-adrenergic receptor binding sites (23%) compared to the ovariectomized group. DHT increased pilocarpine stimulated lacrimal gland fluid secretion (13.26±1.47 μL/min) compared to the ovariectomized group (7.72±0.41 μL/min), but DHT treatment paradoxically decreased basal tear flow rate (1.02±0.04 μL/min) as compared to the ovariectomized rabbits (1.96±0.12 μL/min). DES decreased the total serum T from 59.33±10.54 pg/mL to 21.5±6.06 pg/mL. DES decreased total Na+,K+-ATPase by 12% and increased ß-adrenergic receptor binding sites by 83.3%. These results suggest that androgens play a major role in supporting lacrimal gland secretory function. Additionally, they suggest that estrogens may influence certain aspects of lacrimal functions, although it is not clear to what extent those actions are elicited directly or indirectly.

Hormonal Support of Lacrimal Function, Primary Lacrimal Deficiency, Autoimmunity, and Peripheral Tolerance in the Lacrimal Gland

Several causes of lacrimal insufficiency have been recognized, including Sjögrens syndrome and other immune-related processes as well as a disparate group of non-immune related disorders. However, the mechanisms underlying primary lacrimal deficiency (PLD), the most common cause of dry eye, have remained obscure. After summarizing mechanisms of lacrimal secretion and stimulus-secretion coupling, the authors review the thesis that optimal lacrimal gland function depends on a hormonal milieu in which androgens play a crucial role. According to this thesis, simple acquired PLD results when bioavailable androgen levels decrease below critical values. However, it is noted that PLD also may be complicated by local autoimmune processes, and hypothetical pathways leading to such processes are discussed. Cell death following withdrawal of hormonal support may lead to processing and presentation of parenchymal cell antigens. Normal intracellular membrane traffic patterns may cause acinar cells to secrete autoantigens into the interstitium. When acinar cells have been induced to express major histocompatibility complex Class II molecules, their intracellular membrane traffic may allow them to process and present autoantigens, essentially mimicking the functions of professional antigen presenting cells. The possibility is discussed that perturbations of the spectra of released and presented autoantigens upset the equilibria of idiotypic networks arising to establish peripheral tolerance. The resulting incremental increases in lymphocytic infiltration are suggested to represent essentially cryptic autoimmune processes which may impair lacrimal secretory function and regeneration. Failure to establish peripheral tolerance is predicted to permit unrestrained CD(4) cell proliferation and an environment favoring B cell activation. Recruitment of B lymphocytes, perhaps in events influenced by re-activated viruses, is predicted to lead to Sjögrens autoimmunity as recognized by stringent diagnostic criteria. Finally, the possibility is discussed that androgen supplementation or hormone replacement therapy might prevent simple PLD and avoid the initiation of autoimmune processes.

MHC class II molecules, cathepsins, and La/SSB proteins in lacrimal acinar cell endomembranes

Sjögrens syndrome is a chronic autoimmune disease affecting the lacrimal glands and other epithelia. It has been suggested that acinar cells of the lacrimal glands provoke local autoimmune responses, leading to Sjögrens syndrome when they begin expressing major histocompatibility complex (MHC) class II molecules. We used isopycnic centrifugation and phase partitioning to resolve compartments that participate in traffic between the basolateral membranes and the endomembrane system to test the hypothesis that MHC class II molecules enter compartments that contain potential autoantigens, i.e., La/SSB, and enzymes capable of proteolytically processing autoantigen, i.e., cathepsins B and D. A series of compartments identified as secretory vesicle membranes, prelysosomes, and microdomains of the trans-Golgi network involved in traffic to the basolateral membrane, to the secretory vesicles, and to the prelysosomes were all prominent loci of MHC class II molecules, La/SSB, and cathepsins B and D. These observations support the thesis that lacrimal gland acinar cells that have been induced to express MHC class II molecules function as autoantigen processing and presenting cells.Sjögrens syndrome is a chronic autoimmune disease affecting the lacrimal glands and other epithelia. It has been suggested that acinar cells of the lacrimal glands provoke local autoimmune responses, leading to Sjögrens syndrome when they begin expressing major histocompatibility complex (MHC) class II molecules. We used isopycnic centrifugation and phase partitioning to resolve compartments that participate in traffic between the basolateral membranes and the endomembrane system to test the hypothesis that MHC class II molecules enter compartments that contain potential autoantigens, i.e., La/SSB, and enzymes capable of proteolytically processing autoantigen, i.e., cathepsins B and D. A series of compartments identified as secretory vesicle membranes, prelysosomes, and microdomains of the trans-Golgi network involved in traffic to the basolateral membrane, to the secretory vesicles, and to the prelysosomes were all prominent loci of MHC class II molecules, La/SSB, and cathepsins B and D. These observations support the thesis that lacrimal gland acinar cells that have been induced to express MHC class II molecules function as autoantigen processing and presenting cells.

A Lacrimal Gland is a Lacrimal Gland, But Rodent's and Rabbit's Are Not Human

Research into the physiological processes governing both normal and abnormal functions of the lacrimal gland has used animal models to provide insights that might be applied to improving our understanding of human disease and designing of beneficial therapeutic interventions. Animal models most frequently used are mice, rats, and rabbits. As participants in research into normal and abnormal lacrimal gland function, the authors have observed significant differences between the various animal models, and these differences must be considered in investigational studies. This review summarizes a wide range of topics, including structural organization of the lacrimal gland and the immunological, secretomotor and hormonal processes regulating lacrimal gland function in all three animal models. In addition, comparisons with relevant aspects of the human lacrimal gland are included where permitted by available data.

Sex-dependent parameters related to electrolyte, water and glycoprotein secretion in rabbit lacrimal glands

The lacrimal glands of males and females of various species differ with respect to several morphological, biochemical and functional characteristics. The purpose of this study was to determine the effect of sexual maturation on Na+,K(+)-ATPase, muscarinic cholinergic receptors and beta-adrenergic receptors, which are closely related to the secretion of electrolytes and fluid by the gland, and on other membrane-associated enzymes, specifically galactosyltransferase and alkaline and acid phosphatase. Soluble and total membrane fractions were obtained from lacrimal glands of prepubertal (1.0 kg), pubertal (2 kg), and mature (4 kg) of New Zealand white rabbits of both sexes. Prepubertal and pubertal rabbits exhibited no sex differences in the total amount of lacrimal gland protein or in any of the enzymes or receptors, with the exception of galactosyltransferase and alkaline phosphatase. Galactosyltransferase had higher total and specific activities in prepubertal and pubertal males, and alkaline phosphatase had higher specific activity in prepubertal males. As animals matured, total protein and activities of the enzymes increased, and several quantitative differences between males and females became apparent. Samples from mature females contained significantly less DNA and membrane and total protein. Specific activities of Na+,K(+)-ATPase, cholinergic receptors, galactosyltransferase, and acid and alkaline phosphatase were 40% to 80% greater (p < 0.05) in mature females. Total and specific activity for beta-adrenergic receptors, on the other hand, were higher in the male rabbits. These findings suggest that sex hormones play a role in regulating the levels of expression of a number of enzymes and receptors, including several which are clearly involved in lacrimal secretory functions.

Effects of thyroid hormone on plasma adenosine 3',5'-monophosphate production in man.

Thyroid hormone may nonspecifically modulate cAMP production and end-organ responsiveness to diverse hormonal stimuli. This hypothesis was tested in 18 hyperthyroid, 16 euthyroid, and 8 hypothyroid human subjects by measurement of cAMP in plasma and urine both in the basal state and following stimulation by epinephrine, parathyroid hormone, and glucagon—hormones known to act through cAMP. Supine fasting plasma cAMP (PcAMP) concentrations (mean ± SEM) were minimally elevated in the hyperthyroid patients (23.5 ± 1.3 nM, p < 0.001) when compared with the euthyroid (17.1 ± 0.6 nM) or hypothyroid (20.5 ± 1.7 nM) groups. Infusion of propranolol over 45 min failed to lower basal PcAMP concentrations in 5 hyperthyroid subjects. Epinephrine infusions (0.05 μg/kg/min) caused an exaggerated peak PcAMP response (58.7 ± 5.7 nM) in 5 hyperthyroid patients and a diminished response (27.3 ± 3.2 nM) in 5 hypothyroid patients when compared with 5 euthyroid subjects (42.3 ± 2.6 nM, p < 0.05). Administration of parathyroid hormone, 200 units intravenously, also caused significant differences in urinary cAMP excretion (μmole/hr) in the hyperthyroid (11.37 ± 0.96, p < 0.005) and hypothyroid patients (2.4 ± 0.58, p < 0.001) when compared to the euthyroid group (6.59 ± 0.74). Glucagon (1 mg intravenously) caused an enhanced peak PcAMP response in the hyperthyroid patients (514 ± 34 nM) compared with the euthyroid (240 ± 29 nM) or hypothyroid (223 ± 28 nM) groups (p < 0.005). The PcAMP disappearance half-time following the peak response to glucagon was similar in all three groups, indicating that plasma sampling is probably a valid indicator of cAMP production. These studies demonstrate that thyroid hormone may modulate the response of multiple hormonal effects mediated by cAMP. The findings suggest a further cellular mode of action of thyroid hormone which may account for a number of the metabolic disturbances observed in patients with thyroid disease.

Transcriptional and translational regulation of LH, prolactin and their testicular receptors by hCG and bromocriptine treatments in adult and neonatal rats

Effects of altered gonadotropin and prolactin (PRL) secretion on luteinizing hormone (LH), PRL and their testicular receptors (R) were studied in neonatal and adult rats. Changes in gene expression were monitored by measurements of steady-state mRNA levels. Five-day and 90-day-old male rats received a single s.c. injection of hCG (600 IU/kg), 1 mg/kg bromocriptine (BR) twice daily, or their combination. After 2 or 8 days, the responses of LH, PRL, their testicular R, and testosterone (T) were assessed, including measurements of the appropriate mRNA levels. Vehicle-treated age-matched animals served as controls. hCG suppressed serum LH in 2 days in adult rats from 0.85 +/- 0.16 to 0.04 +/- 0.01 microg/l, and in neonates from 0.59 +/- 0.29 to levels below 0.01 microg/l (p < 0.01 for both). This was accompanied at both ages by a 60% decrease in pituitary content of the LH beta-subunit mRNA (p < 0.01), but a decrease in the alpha-chain (40%, p < 0.05) occurred only in neonates. hCG increased serum PRL in adult rats in 8 days over 2-fold (p < 0.01); this did not occur in neonates. In neonates, BR increased the LH subunit mRNAs 2-fold in 8 days (p < 0.01) without a concomitant effect on serum LH; no BR effects on the LH parameters were seen in adult animals. BR decreased pituitary PRL protein and mRNA levels at both ages (p < 0.01-0.05), but serum PRL decreased only in the adults. The homologous down-regulation of testicular LHR (near 100%) was accompanied in adults by a 30% decrease in LHR mRNA (p < 0.05). Also BR at this age decreased LHR binding (75% in 8 days, p < 0.01), but in this case no change occurred in the cognate mRNA. hCG and BR slightly up-regulated in adults PRLR binding, but only the 2-day effect of BR was accompanied by a 60% increase in PRLR mRNA (p < 0.05). In neonates, both hCG and BR increased testicular LHR and PRLR mRNA levels (p < 0.01-0.05). In adult animals, both hCG and BR suppressed testicular and serum T levels after 8 days (40-70%, p < 0.01-0.05); only BR was inhibitory to T by 8 days in the neonates (p < 0.05). In conclusion, the homologous and heterologous regulatory effects of hCG and BR on LH, PRL and their testicular R levels were only partly explained by changes in steady-state levels of the respective mRNAs. In general, the autoregulatory effects on LHR and PRLR appeared to affect steady-state levels of cognate mRNAs, whereas heteroregulation predominately involved changes at the protein level. The responses of the neonatal pituitary-gonadal axis to hCG and/or BR differed greatly from those observed in the adult, indicating that the mechanisms involved in these regulatory events in adult animals are a result of gradual postnatal development.

Ontogeny of pituitary-gonadal interactions current advances and controversies

The different compartments of the fetal hypothalamic-pituitary-gonadal axis, the hypothalamus, anterior pituitary, and gonads, probably start their embryonic development independently, and become fully interactive as the last link o f their maturation. The developing hypothalamic-pituitary-gonadal axis offers a good model for studies on the mechanisms of regulation of fetal hormonal systems. It is evident that fetal hormonal functions are not the same as those of the adult on a smaller scale, but that there are fundamental differences between the fetus and adult in basic features of the mechanisms o f reproductive hormone action.

ANDROGEN SUPPORT OF LACRIMAL GLAND FUNCTION IN THE FEMALE RABBIT

Dry eye is a major reason for visits to an ophthalmologist’s office. The most probable cause for dry eye is primary lacrimal deficiency (PLD).1 PLD is usually detected in women, most frequently after menopause, during pregnancy or lactation, or when taking estrogen-containing oral contraceptives. These various endocrine states exhibit a complete range of plasma estrogen levels from very low to very high. Thus, plasma estrogen concentrations do not appear to be a common variable in PLD. However, plasma free androgen levels are potentially decreased in all of these states. We have previously demonstrated that ovariectomy of female rabbits2 and hypophysectomy of female rats3 result in a decrease in biochemical correlates of lacrimal gland function. Treatment of these endocrinectomized animals with the potent androgen dihydrotestosterone (DHT) restores the decreases in the biochemical markers of secretion,2,3 specifically, lacrimal gland protein, DNA, Na,K-ATPase, and s-adrenergic receptors. Androgens have been shown to be responsible for the male-like morphological and functional characteristics of the gland, including larger acini,4 greater secretion of IgA,5 and greater production of polymeric IgA receptor, measured as secretory component (SC).6 When female rats are treated with androgens, the morphology of the lacrimal gland changes and resembles the male lacrimal gland.7 However, the major neurotransmitter receptor coupled to secretion in the lacrimal gland, the muscarinic cholinergic receptor, is regulated by circulating levels of prolactin, not androgens.3

Ontogeny of the inhibitory guanine nucleotide-binding regulatory protein in the rat testis: mRNA expression and modulation of LH and FSH action.

The ontogeny of function and mRNA expression of the inhibitory guanine nucleotide-binding regulatory protein (Gi) was studied in the rat testis. Dispersed testis cells of animals aged 8, 15, 20 and 30 days were cultured with or without 100 micrograms/l pertussis toxin (PT) for 24 h. The cells were then cultured for another 24 h with medium only, cholera toxin (CT), PT, or their combination, and the amount of testosterone and cAMP production was measured. PT preincubation increased CT-stimulated cAMP production at all ages, thus indicating the presence of a functional Gi-protein in the postnatal testis. However, when testosterone production was measured, the enhancing effect of PT was absent at the age of 8 days only, indicating that Leydig cells at this age did not have functional Gi-protein. We then cultured 2-day-old and 8-11-day-old testis cells, after 24 h pretreatment with PT, in the presence of ovine follicle-stimulating hormone (FSH) (1 mg/l). The FSH-stimulated cAMP production was enhanced at both ages, thus indicating the presence of a functional Gi-protein in neonatal Sertoli cells. In Northern blot analyses, fetal and postnatal testis tissue had very similar levels of G alpha i2 and G alpha i3 mRNAs; the mRNA level of Gi1 in Northern blots remained low compared to those of Gi alpha 2 and Gi alpha 3. In conclusion, the Gi protein appears in the developing rat testis in utero but the activity first seems to be confined to non-Leydig cells including the Sertoli cell. In Leydig cells, the functional Gi-protein appears between days 8-15 post partum. This finding may be related to the fact that the fetal-neonatal population of Leydig cells possesses a high steroidogenic capacity and an apparent lack of the ability to respond to high gonadotropic stimulation with LH receptor down-regulation and steroidogenic enzyme desensitization.