Rabbit lacrimal acinar cells in primary culture: morphology and acute responses to cholinergic stimulation.

PURPOSE: The rabbit lacrimal gland yields large numbers of viable acinar cells that, when exposed to carbachol, respond with accelerated protein release, fluid phase endocytosis (Lucifer yellow uptake), and Na/H antiport activation. The current study was undertaken to determine whether such cells exhibit similar responses after having been maintained in primary culture. METHODS: Cells were isolated from 2-kg, juvenile male New Zealand White rabbits and maintained in a supplemented DMEM/Ham's F-12 medium for up to 72 hours. RESULTS: Electron microscopy showed the organization of freshly isolated cells to be highly polarized, with secretory vesicles at one pole and nucleus at the other; vesicles were heterogeneous in size and in the electron density of their contents. The cells remained polarized after overnight culture, but the secretory vesicle population was more homogeneous in size and content, and the cells tended to aggregate. After 72 hours, roughly half the cells retained good morphology and cytoplasmic polarization, but the vesicles were enlarged and their contents less electron dense. Cells that had been maintained overnight responded to the addition of 10 microM carbachol with a 32.2% +/- 15.5% (n = 12, P < 0.04) increase in the total amount protein released during a standard 20-minute incubation. This represented a mean 125% increase in the temperature-dependent component of protein release. The protein secretory response was decreased to 14.6% +/- 6.1% (n = 3, P < 0.07) for cells that had been maintained for 72 hours. In the same samples, carbachol increased fluid phase endocytosis by 38.3% +/- 8.1% (P < 0.01) and 70.9% +/- 13.4% (P < 0.025), respectively. The protein secretory response was partially, and the endocytic response fully blocked by 1 mM atropine. CONCLUSIONS: This model could be useful as a simplified system in which to study regulation of acinar cell function over days, rather than hours, as is required in fresh tissue models.

Prandial presbyopia: the muffin man.

Transient blurring of near vision can be due to a variety of causes. We report the case of a 35-year-old man with a 10-year history of blurring of near vision that begins 30 to 45 seconds after he starts to eat and that lasts until 10 to 15 minutes after he stops eating. Magnetic resonance imaging and computed tomography of the brain and orbits did not reveal any abnormality, and stimulation of individual cranial nerves did not result in a loss of near vision. Retinoscopic refraction revealed the loss of 1.5 dioptres of accommodative power in each eye one minute after he began to eat. To the best of our knowledge such blurring of vision at near, immediately after initiating a meal, has not been previously reported. The neuroanatomy of the accommodation and of the gustatory pathways are discussed, as they may relate to this patient's visual complaint.

Electrolyte composition of lacrimal gland fluid and tears of normal and vitamin A-deficient rabbits.

Rabbit tears and lacrimal gland fluid were collected simultaneously during pilocarpine stimulation with the goal of comparing the ionic composition of these fluids at various flow rates. Ions measured were sodium, potassium, calcium, magnesium, zinc, chloride, and bicarbonate. Human tears were also analyzed for purposes of comparison. Generally, tears and lacrimal gland fluid do not differ in ionic composition except for zinc and bicarbonate, which are in higher concentration in tears than in lacrimal gland fluid. The ionic composition of tears and lacrimal gland fluid of vitamin A-deficient rabbits was also analyzed. The maximal flow rate of lacrimal gland fluid was decreased in vitamin A-deficient rabbits as were calcium levels in tears and lacrimal gland fluid, as compared with controls. Concentrations of other ions generally did not differ from normal levels, indicating that vitamin A deficiency has only moderate effects on lacrimal gland function in the rabbit.

The relationship between secretion of retinol and protein by the lacrimal gland.

Secretion of retinol and protein by the rabbit lacrimal gland appear to be closely related, suggesting that they are secreted by the same mechanism. Pilocarpine and vasoactive intestinal peptide (VIP) both stimulate protein and retinol secretion rate in a dose-dependent manner, but the concentrations of retinol and protein in the lacrimal gland fluid are independent of fluid flow at flow rates in excess of 1 microliter/min. Under all conditions of stimulation with pilocarpine and VIP that were studied, the retinol:protein ratio in lacrimal gland fluid of normal rabbits remained constant at 3.3 ng retinol/mg protein. This correlation between retinol and protein secretion by the lacrimal gland suggests that retinol is protein-bound in lacrimal gland fluid. To identify this protein, vitamin A-deficient rabbits were treated orally with 3H-retinyl acetate. Lacrimal gland fluid was collected and analyzed for 3H-retinol and protein. The 3H-retinol in lacrimal gland fluid was identified by reverse-phase HPLC and analysis of protein by gel filtration chromatography showed that this 3H-retinol was associated with protein which eluted from the columns in the 20 kD range.

Tear secretion and lacrimal gland function of rabbits treated with isotretinoin.

Adverse ocular reactions including dry eye symptoms and blepharoconjunctivitis are common side effects of treatment with isotretinoin (13-cis-retinoic acid). However, there is little agreement in the literature on the effect of this drug on the tears. Because we have previously shown that the lacrimal gland secretes isotretinoin, we conducted a study of the effect of isotretinoin on lacrimal gland function. Rabbits were treated with isotretinoin for 5 months. Throughout the study tear secretion was monitored by the Schirmer test. At the end of the study lacrimal gland function was assessed by measurement of fluid and protein secretion rates and secretion of retinol in response to a pilocarpine stimulus. Lacrimal gland function was not affected by isotretinoin as compared with a group of age-matched control rabbits; however, Schirmer test scores were significantly increased in the treated animals as compared with control values. We conclude that isotretinoin is not toxic to the lacrimal gland of rabbits. This suggests that ocular irritation in patients treated with isotretinoin is not caused by decreased tear secretion during therapy.

Treatment of corneal xerophthalmia in rabbits with micromolar doses of topical retinoic acid.

Several reports have appeared on the efficacy of topically applied 0.01% or 0.1% all-trans retinoic acid (0.04-0.4 millimolar) for treatment of xerophthalmia, conjunctival squamous metaplasia, and corneal epithelial erosions in humans and animals. An observation common to many of these studies is the occurrence of an adverse reaction to retinoic acid in the form of lid margin hyperemia and blepharoconjunctivitis. Since retinoic acid is biologically active at micromolar to nanomolar concentrations, it may be possible to reduce side effects while maintaining therapeutic effectiveness by reducing the retinoic acid concentration in ophthalmic formulations. In the present study, topical 0.005% retinoic acid in petrolatum ointment reversed corneal keratinization in xerophthalmic, vitamin A-deficient rabbits in 3-4 days while 0.0005% (2 micromolar) retinoic acid ointment was effective in 4-6 days. Further clinical trials of topical retinoic acid for treatment of ocular surface disease should be conducted using micromolar concentrations of retinoic acid which are expected to maintain a therapeutic effect while reducing adverse reactions.

Isotretinoin in lacrimal gland fluid and tears.

Isotretinoin (13-cis-retinoic acid) is used in the treatment of severe cystic acne. Adverse ocular reactions, including blepharoconjunctivitis and dry eye symptoms, are frequent side effects of this drug. Our previous observation that retinol is present in tears and lacrimal gland fluid suggests that isotretinoin may also be secreted by the lacrimal gland. Rabbits were treated with isotretinoin, and lacrimal gland fluid was collected from the cannulated lacrimal gland duct. Tears were collected from patients who were being treated with isotretinoin. Lacrimal gland fluid and tears were analyzed by reverse-phase high-pressure liquid chromatography and a peak eluted from each sample, which was identified as isotretinoin. We conclude that the lacrimal gland is able to secrete isotretinoin in addition to retinol and that, in animals and patients treated systemically with isotretinoin, the ocular surface is exposed to the drug via the tear film.

Retinol secretion by the lacrimal gland.

In order to determine the source of the retinol which has been identified in the tear fluid, the lacrimal gland ducts of rabbits and rats were cannulated and the collected lacrimal gland fluid was analyzed by high performance liquid chromatography. Retinol was identified in the lacrimal gland fluid of rabbits and rats, and it is concluded that the lacrimal gland is the source of retinol in the tears. Dose-response studies show that intravenously administered pilocarpine and intra-arterial acetylcholine stimulate secretion of retinol by the lacrimal gland. Intravenous administration of vasoactive intestinal peptide (VIP) also stimulates retinol secretion in a dose-response manner. These observations are similar to the effects of cholinergic drugs and VIP on protein secretion by the lacrimal gland.