Gi/Go couple met-enkephalin to inhibition of cholinergic and beta-adrenergic stimulation of lacrimal secretion.

PURPOSE: To determine whether G-protein-mediated inhibition of secretion by met-enkephalin involves cyclic adenosine monophosphate (cAMP)-dependent events and to identify the G proteins that couple met-enkephalin to inhibition of lacrimal secretion. METHODS: Secretion of protein was measured in 3-day primary cultures of rabbit lacrimal acini exposed to vehicle, the cholinergic agonist carbachol (Cch), the beta-adrenergic agonist isoproterenol (Isop), vasoactive intestinal peptide (VIP), or forskolin (FSK) with or without the enkephalin analog D-ala2-met-enkephalinamide (DALA). In separate experiments, cells were pretreated with pertussis toxin or polyclonal antibodies against the alpha subunits of Gi/Go to determine the physiologic role of G proteins in met-enkephalin inhibition of the release of lacrimal protein. Adenylyl cyclase (AC) activity was measured by a cAMP-dependent protein kinase binding assay in lacrimal membranes in response to the same agonists used in the secretion studies. RESULTS: Cch resulted in a significant increase in protein release from cultured lacrimal acini. Increased secretion also occurred with Isop, VIP, and FSK. Cch- and Isop-stimulated secretion was inhibited by DALA to near-basal values. However, DALA did not inhibit VIP- or FSK-stimulated secretion. The inhibitory effect of DALA on Cch and Isop stimulation of secretion was reversed by pertussis toxin. Inhibition of Cch-stimulated secretion was blocked by antibody specific to a common peptide sequence of Gialpha1 and Gialpha2 but was not blocked by anti-Gialpha1 antibody. The inhibitory effect on Cch-stimulated secretion was also blocked by anti-Gialpha3 and anti-Goalpha. Similar experiments resulted in a reversal of DALA inhibition of beta-adrenergic stimulation of secretion by immunoneutralization of Gialpha1/2 and Goalpha but not by immunoneutralization of Gialpha1 or Gialpha3. VIP, Isop, and FSK significantly stimulated AC. However, Cch had no effect on the activity of the enzyme. In addition, DALA had no effect on AC activity under any conditions. CONCLUSIONS: These results show that enkephalin inhibition of cholinergic and beta-adrenergic stimulation of secretion is mediated by Gi2, Gi3, and Go. The effector coupled by the G proteins is not AC. However, we suggest a role for met-enkephalin in G-protein-coupled modulation of ion channels important for cholinergic and beta-adrenergic stimulation of lacrimal secretion.

Adrenergic stimulation of lacrimal protein secretion is mediated by G(q/11)alpha and G(s)alpha.

PURPOSE: The intent of this work was to continue the characterization of G protein coupling of receptors to lacrimal secretion by determination of whether the alpha subunits of the heterotrimeric G proteins G(q/11) and G(s) couple alpha and beta-adrenergic receptor activation to stimulation of protein secretion by isolated lacrimal acini. In addition, we assessed the possibility that the G beta gamma dimer influences stimulated lacrimal protein secretion. METHODS: Primary cultures of rabbit lacrimal acini were permeabilized by streptolysin-O (SLO) to allow cellular insertion of polyclonal antibodies to G(q/ll)alpha, G(s)alpha or G beta or GDP beta S. Following this, secretion of exocytotic protein was measured in response to vehicle, the alpha(1)-adrenergic agonist phenylephrine, the beta-adrenergic agonist isoproterenol, the cholinergic agonist carbachol or vasoactive intestinal peptide (VIP). RESULTS: Phenylepherine and isoproterenol resulted in a significant increase in protein release from cultured lacrimal acini. The increase in secretion elicited by the adrenergic agonists, however, was much less than that induced by either carbachol or VIP. Antibody to G(q/11)alpha blocked phenylephrine stimulated secretion 32% but had no effect on isoproterenol or VIP stimulation of secretion. In contrast, the same antibody blocked carbachol stimulated secretion by 72%. Antibody to G(s)alpha blocked isoproterenol stimulated secretion 20%, had no effect on phenylephrine stimulation, blocked carbachol stimulation by 27% and VIP stimulation by 69%. Antibody to G beta did not effect stimulation of secretion by any agonist. The degree of inhibition of secretion following exposure to GDPbetaS did not exceed that obtained with the G protein subunit antibodies. CONCLUSIONS: The alpha subunit of G(q/11) couples activation of alpha(1)-adrenergic receptors to exocytotic release of protein in the lacrimal gland. Activation of beta-adrenergic receptors does not involve G(q/11) but is mediated by the alpha subunit of G(s). G protein coupling of the adrenergic receptors to secretion appears to be limited compared to cholinergic and VIP stimulation and might suggest the occurrence of the activation of intracellular signaling pathways independent of receptor-G protein-effector regulation of adrenergic stimulation of lacrimal secretion.

Gi2 and Gi3 couple met-enkephalin to inhibition of lacrimal secretion.

PURPOSE: The intent of this study was to identify the pertussis toxin-sensitive G proteins that couple met-enkephalin to the inhibition of cholinergically stimulated secretion in rabbit lacrimal gland acini. METHODS: The authors detected G proteins in membranes from freshly isolated glands, freshly isolated acini, and cultured lacrimal acini from rabbits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antibodies against the alpha subunits of Gi1, Gi1 and Gi2, or Gi3 were used in cultured acini permeabilized by streptolysin-O to determine the role of the G proteins in met-enkephalin inhibition of cholinergic stimulation of lacrimal acinar protein release. RESULTS: Western blot analysis showed the presence of the alpha subunits of Gi2 and Gi3, but not Gi1, in all three membrane preparations. The met-enkephalin analog D-Ala2-methionine enkephalinamide (DALA) inhibited cholinergic stimulation of secretion by cultured rabbit acinar cells to near basal levels. Inhibition of secretion by DALA was blocked by insertion of antibody to a peptide sequence common to Gialpha1 and Gialpha2, but was not blocked by antibody against a specific Gialpha1 sequence. The inhibitory effect of DALA also was blocked by antibody to a Gialpha3 sequence. At low doses of anti-Gialpha1/2 and anti-Gialpha3 in combination, the effect on reversal of inhibition was additive. However, at higher doses, the effect of the combination was no greater than the effect of either antibody alone. CONCLUSIONS: These results demonstrate that met-enkephalin inhibition of cholinergic secretion is mediated by way of the pertussis toxin-sensitive G proteins Gi2 and Gi3 in cultured rabbit lacrimal acini. Because the effects of the G proteins are not additive, the intracellular events distal to G protein activation most likely converge at some point before exocytosis.

Effect of sensory denervation on the structure and physiologic responsiveness of rabbit lacrimal gland.

PURPOSE: This work was conducted to determine the effects of unilateral trigeminal ganglion ablation on lacrimal gland structure and secretory activity. METHODS: Adult male New Zealand rabbits underwent unilateral thermocoagulation of the ophthalmic division of the trigeminal ganglion. Sensory denervation was affirmed by anatomic inspection of the lesion and transmission electron microscopy (TEM) of the lacrimal gland innervation. Eight to 10 days after the procedure, the intraorbital lacrimal glands were removed from both sides. To compare the physiologic competence of the intact and denervated glands, freshly isolated gland fragments from the paired intact and denervated glands were stimulated with carbachol (100 microM), isoproterenol (10 microM), phorbol-12,13-dibutyrate (PDBu, 10 microM), forskolin (40 microM), or vehicle. Total secreted protein was measured at 30 or 60 min after the establishment of baseline values. Intact and denervated glands also were examined by light and TEM, and the morphologic appearance of the acinar structures as well as the appearance of nerves innervating the gland after denervation were assessed. Similar experiments were conducted with animals that underwent unilateral superior cervical ganglionectomy. RESULTS: Tissues from sensory denervated glands released significantly more protein than did tissues from innervated glands in response to in vitro stimulation by carbachol or isoproterenol but not in response to PDBu or forskolin. Microscopy showed that the acinar cells that had undergone sensory denervation showed a massive accumulation of secretory granules. The secretory granules filled the entire cytoplasmic space and displaced the ellipsoidal nuclei to the extreme periphery. Examination of segments of nerves revealed numerous unmyelinated axons, a few small-diameter myelinated axons, and a large amount of nerve degeneration after sensory denervation. In contrast to the effects of sensory denervation, sympathetic denervation did not alter either the acinar appearance or secretory responsiveness of the gland. CONCLUSION: Loss of the considerable sensory innervation from the trigeminal ganglion has pronounced effects on the pharmacologic responsiveness and the structure of the lacrimal gland. The effects of sensory innervation on the gland may be mediated through two possible pathways: direct input to the gland or control of the preganglionic parasympathetic pathway.

Gs and Gq/11 couple vasoactive intestinal peptide and cholinergic stimulation to lacrimal secretion.

PURPOSE: The intent of this study was to determine the physiological role of selected G proteins in receptor-mediated protein release by lacrimal acini. METHODS: The role of G proteins in lacrimal secretion was determined in tissues obtained from the lacrimal glands of adult male New Zealand White rabbits. Pertussis toxin treatment of primary acinar cultures and permeabilization of cultured acini with streptolysin-O and insertion of GDP beta S or antibodies against the alpha subunit of Gs or Gq/11 were used to determine the role of G proteins in vasoactive intestinal peptide (VIP) and carbachol-stimulated lacrimal secretion. Gs and Gq/11 were identified in lacrimal membranes obtained from freshly isolated lacrimal gland fragments, freshly isolated acini, and cultured acini by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. RESULTS: Permeabilization by streptolysin-O and introduction of guanosine thiodiphosphate into cultured acini blocked stimulation of protein released by either 100 nM VIP or 100 microM carbachol by approximately 50%. Exposure of cultured acini to 100 ng/ml pertussis toxin for 36 to 48 hours did not affect stimulated release by either agonist, indicating that the guanosine triphosphate-dependent actions of VIP and carbachol are mediated through pertussis toxin-insensitive G proteins. Pertussis toxin-insensitive G proteins in lacrimal membranes obtained from freshly isolated glands, freshly isolated acini, and cultured acini were identified with polyclonal antibodies to the alpha subunits of Gs and Gq/11. Immunoblotting of lacrimal membranes with anti-Gs alpha antiserum showed two immunoreactive bands at 44 and 47 kDa. Anti-Gq/11 alpha antiserum detected a single band at 46 kDa in similar membrane preparations. Anti-Gs alpha antiserum reduced the secretory response to VIP by 64% and to carbachol by 37%. Introduction of anti-Gq/11 alpha antiserum reduced the response to carbachol by 70%; however, the response to VIP was unchanged. Simultaneous introduction of both antisera caused no further reduction of VIP-stimulated release than did anti-Gs alpha antiserum alone. However, simultaneous introduction of both anti-Gs alpha and anti-Gq/11 alpha antisera resulted in complete inhibition of the effects of carbachol on protein release by cultured acini. CONCLUSIONS: These results show that VIP receptor activation of lacrimal protein release is mediated through Gs, whereas cholinergic stimulation involves both Gs and Gq/11. From the authors' results, the authors conclude that Gs links VIP receptor activation to adenylyl cyclase and cyclic adenosine 3'-5' monophosphate production and the ultimate release of protein by acinar cells and that Gq/11 links muscarinic receptor activation to phospholipase C and IP3 and diacylglycerol accumulation, which also leads to protein release. Furthermore, it is hypothesized that Gs has an additional role in the regulation of vesicular traffic and exocytosis.

Identification of G proteins in lacrimal gland.

PURPOSE: The intent of this study was to identify and characterize guanine nucleotide binding proteins (G proteins) that are a component of cell signaling of stimulus-secretion coupling in lacrimal gland. METHODS: Membranes were isolated from the lacrimal glands of male Sprague-Dawley rats and New Zealand rabbits and were used to identify G proteins in lacrimal gland by Western blot analysis. Solubilized membrane proteins from lacrimal glands and from a positive tissue control (SH-SY5Y cells) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha subunits of the G proteins were identified by immunoreactivity, with specific peptide directed antisera against Gs alpha, Gi alpha 1, Gi alpha 1/2, and Go alpha. The initial characterization of coupling of specific G proteins to receptors was accomplished by preincubation of membranes with nonimmune serum, anti-Gs alpha, or anti-Go alpha antiserum and assay of adenylyl cyclase activity in the presence of the neuropeptide, vasoactive intestinal peptide (VIP), or forskolin. RESULTS: Gs alpha, Gi alpha 3, and Go alpha were present in all three membrane preparations. Gs alpha subunits were detected as two bands at 45 to 48 kd and 48 to 54 kd. Gi alpha 3 was detected as a single protein at 40 to 41 kd, and at least one form of Go alpha with a molecular weight of 40 kd was detected in all three preparations. Gi alpha 1 was detected in immunoblots of rat lacrimal and SH-SY5Y membranes at 41 kd, and the density of the band was enhanced in blots probed with anti-Gi alpha 1/2 antiserum. Immunoreactivity to anti-Gi alpha 1 or anti-Gi alpha 1/2 was faint or not detectable in rabbit lacrimal membranes. A prominent band was detected in rabbit and rat lacrimal but not in SH-SY5Y membranes at 31 kd with anti-Gi alpha 1/2 antiserum, which may represent a G protein involved in exocytosis. Coupling of VIP receptor activation to adenylyl cyclase by Gs alpha was evidenced by the reduction of VIP stimulation of the enzyme by preincubation of rabbit membranes with anti-Gs alpha antiserum. In contrast, preincubation of membranes with anti-Go alpha antiserum resulted in an increase in activity of adenylyl cyclase in the presence of VIP. CONCLUSIONS: Detection of specific alpha subunits in lacrimal gland indicates that Gs, Gi, and Go are present in rabbit and rat lacrimal gland. Both Gs and Go influence VIP stimulation of lacrimal adenylyl cyclase and thus are presumed to be involved in signal transduction, leading to second-messenger accumulation and subsequent regulation of lacriminal function, including secretion. In addition, an unidentified protein is present in lacrimal gland that may represent one of the guanine nucleotide binding proteins involved in exocytosis.

Morphology and physiologic responsiveness of cultured rabbit lacrimal acini.

PURPOSE: To assess the morphology and physiologic response of intact rabbit lacrimal acini maintained in culture for as long as 14 days and to compare the characteristics of the cultured acini with untreated fresh lacrimal gland, incubated lacrimal tissue slices, and freshly isolated acini. METHODS: Acini were obtained by enzymatic digestion of lacrimal glands of adults male New Zealand white rabbits and cultured for as long as 14 days on Matrigel-coated inserts in DMEM-F12. Untreated fresh lacrimal gland, incubated lacrimal tissue slices, freshly isolated acini, and intact cultured acini were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Maintenance of physiologic responsiveness of the cultured acini was assessed by the comparison of the release of protein in response to muscarinic cholinergic, alpha 1- and beta-adrenergic, and peptidergic stimulation of the cultured acini with the secretory response of incubated tissue slices. RESULTS: As examined by TEM, the ultrastructure of intact acini cultured for 3 and 7 days was notably similar to fresh tissue and incubated tissue slices. Recovery of the cultured acini from perturbations induced by the digestion procedure resulted in acini that were firmly attached to the filter supports and that exhibited histotypic acinar morphology, including lumenal microvilli, apically situated secretory granules and junctional complexes, lateral desmosomes, and appropriate secretory organelles. Secretory granules and associated organelles also were prominent in 14-day cultures; however, by day 14, as visualized by SEM, the acini had flattened. TEM of acini at this time showed that they had lost much of their acinar organization and cellular polarization. Outgrowths composed of acinar cells could be observed by day 7 by both TEM and SEM, and SEM demonstrated non-acinar cells growing on the filter surface in 14-day cultures. The morphologic and physiologic response of the acinar cells to autonomic agonists was similar in incubated tissue slices and cultured acini. Morphologically, acinar cells in both preparations responded to cholinergic stimulation by releasing secretory granules, resulting in honeycombed regions in the cell. Assay of secreted protein in response to cholinergic, alpha 1- and beta-adrenergic, and peptidergic stimulation indicated that 3-day cultures of acini retain the response to carbachol, phenylephrine, and vasoactive intestinal peptide. However, the response to isoproterenol is diminished when compared with incubated tissue slices. CONCLUSIONS: Under the conditions described, cultures of intact lacrimal acini from New Zealand white rabbits maintain histotypic morphology and physiologic responsiveness similar to fresh lacrimal gland and incubated tissue slices. Culture of the acini affords a useful alternative for the study of chronic, as well as acute, effects of neurotransmitters, neuropeptides, hormones, and cytokines on the structure and function of the gland.