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The role of HCO3 - transport in relation to fluid secretion by submucosal glands is being studied in sheep, pigs, cats and humans. Optical methods have been developed to measure secretion rates of mucus volume from single glands with sufficient temporal resolution to detect differences in minute-by-minute secretion rates among glands. The ionic composition and viscoelastic properties of the uncontaminated gland mucus are measured with a combination of ratiometric fluorescent indicators, ion-selective microelectrodes, FRAP, and a miniaturized, magnetic force viscometer. Sheep glands secreted basally at low rates, showed small, transient responses to alpha- and beta-adrenergic agonists, and large responses to a cholinergic agonist, carbachol. Peak rates and temporal patterns of responses to carbachol differed markedly among glands. To assess the contribution of HCO3 - transport to gland secretion, we either inhibited Na+ /K+ /2Clcotransporter (NKCC) with bumetanide or replaced HCO3 - with HEPES and gassed with O2. Bumetanide caused a small, non-significant inhibition of basal secretion, but removal of HCO3 - /CO2 significantly reduced basal secretion almost by half. Both bumetanide and removal of HCO3 - /CO2 reduced carbacholstimulated secretion significantly, with HCO3 - removal having the larger effect: a reduction to 33% of control (P<0.01). The remaining secretory response to carbachol was nearly eliminated by bumetanide. Sheep mucus pH measured with ion selective electrodes was about 0.4 log more acidic than the bath. In humans, we observed the same pattern of responses to agonists and antagonists as in sheep, and observed a mucus pH of 7.0 using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We hypothesize that HCO3 - transport is important in the formation of mucus secretion, but that most HCO3 - is scavenged before the final mucus appears at the duct opening.