Morphological and Functional Changes in Cell Junctions During Secretory Stimulation in the Perfused Rat Submandibular Gland

Abstract

To examine the influence of cholinergic and beta-adrenergic agents on paracellular transport, we applied confocal microscopy and freeze-fracture to the isolated, perfused submandibular gland of the rat. By confocal microscopy, perfusion of lucifer yellow through an arterial catheter, revealed a bright fluorescence in the basolateral spaces of acini, but not in the intercellular canaliculi. However, addition of isoproterenol on carbachol stimulation, induced lucifer yellow fluorescence in intercellular canaliculi. This finding indicates that isoproterenol is capable of opening the paracellular route. The tight junction strands surrounding intercellular canaliculi were visualized using freeze replicas. Fixation was carried out both by vascular perfusion with Karnovsky's solution and by metal contact rapid freezing with liquid helium. In the chemically-fixed specimens, the strand particles of tight junctions formed 2-5 lines at the P-face along most of the apical portion at rest. With carbachol/isoproterenol stimulation, the strand particles rearranged with free ends and terminal loops. In the rapidly frozen specimens, the strand particles were arranged more irregularly even in the resting state. The meshwork of strands became more disheveled and interrupted during carbachol/ isoproterenol stimulation. The present findings led us to conclude that: 1) the beta-adrenergic agent, isoproterenol, can open the paracellular transport. 2) in the rapidly frozen specimen, the tight junction strand particles are arranged roughly and become disheveled and interrupted during stimulation by carbachol/isoproterenol. These findings may be related to rearrangement of subcellular structures, especially of the actin filament network.

Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals

“…7E). Isoproterenol has been previously described to transiently modify the permeability of the tight junctions up to 6 h when injected into the rat SMGs (16,25). To determine whether the increase in pVenus expression could be linked to this phenomenon, rather than to compensatory endocytosis, we injected isoproterenol and then administered the plasmid DNA after 1, 4, and 8 h. At 16 h after the DNA injection, the glands were imaged, and pVenus was expressed in cells only when isoproterenol was administered together with the plasmid DNA (not shown).…”

“…C: time course of pVenus expression in SMGs. Plasmid DNA was injected with 0.25 mg/kg isoproterenol (pVenus ϩ ISOP) or saline (pVenus), as described above, and SMGs were imaged at 8,16,30,48, and 72 h after injection to count the number of cells expressing pVenus. D: stimulation of muscarinic and adrenergic receptors.…”

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells

“…In the isolated rat submandibular gland perfusion model, both carbachol and pilocarpine significantly increase the transport of fluorescently labeled tracers (e.g. Lucifer Yellow) from basolateral spaces of acini into intercellular canaliculi; in rapidly frozen specimens, tight junction strands as well as actin filament network, are rearranged roughly and become interrupted after carbachol stimulation (Segawa, 1994;Hashimoto et al, 2003;Hashimoto and Murakami, 2009). These data suggest that epithelial tight-junction-based paracellular transport is also controlled by the cholinergic system.…”

“…In MDCK cells, the mAChR agonist carbachol inhibits the pertussis-toxin-induced decrease in paracellular permeability (Balda et al, 1991). In rat submandibular epithelium, carbachol or pilocarpine increases paracellular transport and causes morphological changes in tight junction structure (Segawa, 1994;Hashimoto et al, 2003). These observations suggest that mAChR activation might also regulate epithelial material transport through paracellular pathway.…”

The potential of functional MRI as a biomarker in early Alzheimer's disease

“…Potential mechanisms that may contribute to this phenomenon include cholinergic or other neurotransmitter upregulation (DeKosky, et al, 2002); aberrant sprouting of cholinergic fibers (Hashimoto, et al, 2003), inefficiency in synaptic transmission (Stern, et al, 2004), increased calcium influx or excitotoxicity (Busche, et al, 2008, Palop, et al, 2007), or alterations in glutamatergic receptor (Rammes, et al, 2011). Further research to determine the specificity of hyperactivation to stage of disease and task performance, the relationship to baseline perfusion and metabolism, and the association with imaging markers of molecular pathology, including amyloid deposition and neurotransmitter systems, is clearly needed to elucidate this phenomenon.…”