We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP] i (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n ‫؍‬ 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP] i suggests that altered secretion of gland mucus could contribute to CF lung disease.

Wine. Optical method for quantifying rates of mucus secretion from single submucosal glands. Am J Physiol Lung Cell Mol Physiol 281: L458-L468, 2001.-We describe an optical method to quantify singlegland secretion. Isolated tracheal mucosa were mounted at the air-Krebs interface and coated with oil. Gland secretions formed spherical bubbles that were digitally imaged at intervals, allowing rates of secretion to be calculated. We monitored 340 glands in 54 experiments with 12 sheep. Glands secreted basally at low rates (0.57 Ϯ 0.04 nl ⅐ min Ϫ1 ⅐ gland Ϫ1 , 123 glands) in tissues up to 9 h postharvest and at lower rates for up to 3 days. Carbachol (10 M) stimulated secretion with an early transient and a sustained or oscillating phase. Peak secretion was 15.7 Ϯ 1.2 nl ⅐ min Ϫ1 ⅐ gland Ϫ1 (60 glands); sustained secretion was 4.5 Ϯ 0.5 nl ⅐ min Ϫ1 ⅐ gland Ϫ1(10 glands). Isoproterenol and phenylephrine (10 M each) stimulated only small, transient responses. We confirmed that cats have a large secretory response to phenylephrine (11.6 Ϯ 3.7 nl ⅐ min Ϫ1 ⅐ gland Ϫ1 , 12 glands), but pigs, sheep, and humans all have small responses (Ͻ2 nl⅐ min Ϫ1 ⅐ gland Ϫ1 ). Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO 3 Ϫ replacement with HEPES, and 92% by both. The distribution of secretion rates was nonnormal, suggesting the existence of subpopulations of glands.

Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl -and HCO 3 -, and clotrimazole sensitive. Loss of "housekeeping" gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.

The induction of synaptic plasticity is known to be influenced by the previous history of the synapse, a process termed metaplasticity. Here we demonstrate a novel metaplasticity in which group I metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) of synaptic transmission is regulated by previous mGluR activation. In these studies, the group I mGluR-dependent LTD induced by the selective agonist (RS)-3,5-dihydroxyphenylglycine (DHPG-LTD) was inhibited by previous preconditioning brief high-frequency stimulation (HFS), regardless of whether the preconditioning HFS induced long-term potentiation. Blockade of NMDA receptors during the preconditioning HFS did not alter the inhibition of DHPG-LTD by the HFS. However, antagonism of mGluRs during the preconditioning HFS did prevent the inhibition of DHPG-LTD by the HFS. In addition, blocking PKC stimulation during the preconditioning HFS also prevented the inhibitory effect of HFS on DHPG-LTD. The DHPG-LTD itself was not inhibited by blocking PKC stimulation but was inhibited by blocking the p38 mitogen-activated protein kinase (MAPK) pathway. Thus, whereas the DHPG-LTD is mediated via activation of the p38 MAPK pathway, the inhibitory effects of preconditioning HFS on DHPG-LTD are mediated via stimulation of group I/II mGluRs, activation of PKC, and subsequent blocking of the functioning of group I mGluR.

Autophosphorylation of α‐Ca2+/calmodulin kinase II (αCaMKII) at Thr286 is thought to be a general effector mechanism for sustaining transcription‐independent long‐term potentiation (LTP) at pathways where LTP is NMDA receptor‐dependent. We have compared LTP at two such hippocampal pathways in mutant mice with a disabling point mutation at the Thr286 autophosphorylation site. We find that autophosphorylation of αCaMKII is essential for induction of LTP at Schaffer commissural–CA1 synapses in vivo, but is not required for LTP that can be sustained over days at medial perforant path–granule cell synapses in awake mice. At these latter synapses LTP is supported by cyclic AMP‐dependent signalling in the absence of αCaMKII signalling. Thus, the autophosphorylation of αCaMKII is not a general requirement for NMDA receptor‐dependent LTP in the adult mouse.

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1. The whole‐cell patch clamp technique was used to investigate the swelling‐activated currents in bovine non‐pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB). The I‐V relationship was shifted in the direction expected for a Cl‐ current when the external Cl‐ was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid (SITS) and 4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid (DIDS), also inhibited this current. 5. The volume‐activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time‐dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl‐ current. 8. An antibody (C219) to the MDR1 gene product, P‐glycoprotein, caused a functional block of the swelling‐activated Cl‐ current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB‐1 demonstrated the presence of P‐glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non‐pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)‐independent, ATP‐dependent Cl‐ current and that the activity of this current is associated with P‐glycoprotein. 11. It is suggested that this Cl‐ current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.

1. The induction of long-term potentiation (LTP) was investigated in the rat dentate gyrus in the presence of ryanodine, an agent which is known to selectively bind to the ryanodine receptor (RyR) Ca2+ channels which regulate Ca2+ release from intracellular Ca2+ stores. 2. In control media, high frequency stimulation (HFS) induced LTP, and prolonged low frequency stimulation (LFS) induced long-term depression (LTD), of field excitatory postsynaptic potentials (EPSPs) and patch clamped excitatory postsynaptic currents (EPSCs).3. In the presence of ryanodine, at a threshold concentration of about 1 uM, HFS-induced LTP was inhibited, whereas LFS (5 Hz, 900 pulses) now induced LTP.