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We have previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) results in potentiation of N-methyl-D-aspartate-induced currents (I(NMDA))of receptors contained in primary cultured cerebellar granule cells (CGCs). The purpose of this study was to identify which PKC isoform(s) was responsible for this effect by using the whole-cell patch-clamp technique. Experiments were conducted on CGCs that expressed both the NR2A and NR2B NMDA receptor subunits as well as the PMA-sensitive PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma, and . As observed previously, N-methyl-D-aspartate-induced peak currents (I(Pk)) were enhanced by a 12.5-min, 100 nM PMA exposure at 37 degrees C under normal recording conditions. Potentiation of receptor function was not observed when extracellular Ca(2+) was removed and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was present inside the cell. PMA-induced potentiation of I(Pk) did not occur when PKCalpha-specific antibody was introduced into the cell via the recording electrode. However, in similar experiments with antibodies specific for PKCbetaII, delta, epsilon, gamma, and , PMA potentiation of I(Pk) was observed. Down-regulation of PMA-sensitive PKC isoforms by an overnight exposure of 100 nM PMA resulted in lack of potentiation by PMA that was rescued when catalytically active PKCalpha was introduced into the cell via the patch electrode. PMA potentiation of I(Pk) was not recovered when catalytically active PKCbetaI, PKCbetaII, or PKCgamma was introduced into the cell via the patch electrode. Collectively, our data provide strong evidence that PMA-enhanced function of native NMDA receptors expressed in primary cultured CGCs is mediated by activation of PKCalpha.

Background Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA. Results We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or aprA - cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA - cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a K D of 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA - cells with a K i of 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA. Conclusion Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA - cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.

Continuous isometric microfocal X-ray computed tomography (CT) scans were acquired from an AKR/J mouse, Brown-Norway rat, and Hartley guinea pig. The anatomy and volume of the paranasal sinus cavities were defined from 2-dimensional (2-D) and 3-dimensional (3-D) CT images. Realistic 3-D images were reconstructed and used to determine the anterior maxillary, posterior maxillary, and ethmoid sinus cavity airspace volumes (mouse: 0.6, 0.7, and 0.7 mm(3), rat: 8.6, 7.7, and 7.0 mm(3), guinea pig: 63.5, 46.6 mm(3), and no ethmoid cavity, respectively). The mouse paranasal sinus cavities are similar to the corresponding rat cavities, with a reduction in size, while the corresponding maxillary sinus cavities in the guinea pig are different in size, location, and architecture. Also, the ethmoid sinus cavity is connected by a common drainage pathway to the posterior maxillary sinus in mouse and rat while a similar ethmoid sinus was not present in the guinea pig. We conclude that paranasal sinus cavity airspace opacity (2-D) or volume (3-D) determined by micro-CT scanning may be used to conduct longitudinal studies on the patency of the maxillary sinus cavities of rodents. This represents a potentially useful endpoint for developing and testing drugs in a small animal model of sinusitis.

A novel series of cyclobutenedione centered C(4)-alkyl substituted furanyl analogs was developed as potent CXCR2 and CXCR1 antagonists. Compound 16 exhibits potent inhibitory activities against IL-8 binding to the receptors (CXCR2 Ki=1 nM, IC(50)=1.3 nM; CXCR1 Ki=3 nM, IC(50)=7.3 nM), and demonstrates potent inhibition against both Gro-alpha and IL-8 induced hPMN migration (chemotaxis: CXCR2 IC(50)=0.5 nM, CXCR1 IC(50)=37 nM). In addition, 16 has shown good oral pharmacokinetic profiles in rat, mouse, monkey, and dog.

Introduction: A method to measure the mucin concentration in bronchoalveolar lavage (BAL) fluid was developed to aid efforts to identify pharmacologically the mechanisms that modulate pathophysiological mucin secretion. Mucins are the major macromolecular components of mucus. In the airways, mucus is the first line of defense against inhaled microorganisms (infection) and particulates (irritation). Methods :A n enzyme-linked immunosorbent assay (ELISA) was developed, comparing two monoclonal anti-mucin antibodies (A10G5 and 45M1) raised to human mucin, to quantify the mucin in BAL fluid from animal models of pulmonary inflammation. To validate the ELISA method, rats were exposed to ovalbumin (OVA, in sensitized rats), lipopolysaccharide (LPS), vanadium pentoxide (V2O5), or saline. One hundred microliters of BAL fluid was analyzed for mucin concentration. Pooled BAL fluid from untreated rats was used as an internal bplate standardQ, as a standard mucin that cross-reacts with A10G5 was unavailable. Results: We found both antibodies reacted with rat, human, and guinea-pig mucin; where the 45M1 antibody also reacted with the mucin in porcine BAL, while A10G5 did not. We determined the mucin concentration in each BAL fluid sample relative to the standard, defined as a mucin concentration of 100 plate units. BAL fluid from LPS (218F25 plate units, n=5), OVA (386F31, n=3), V2O5 (1208F450, n=6) challenged rats displayed significantly elevated mucin concentration over their saline controls (126F22, n=12). Subsequently, the 45M1 antibody displayed immunoreactivity with a commercially available crude preparation of porcine stomach mucin, allowing us to calculate the concentration of mucin directly compared to the known concentration of the porcine stomach mucin standard. Both the 45M1 and A10G5 based ELISA assays detected higher mucin content in the saline challenged rat than the saline challenged guinea pig BAL. Discussion: The recent availability of the 45M1 antibody and the use of the crude purification of porcine stomach mucin as a reference standard should allow for direct comparison of mucin concentration in BAL (and other fluids).

Submucosal glands secrete macromolecules and liquid that are essential for normal airway function. To determine the mechanisms responsible for airway gland secretion and the interaction between gland secretion and epithelial ion transport, studies were performed in porcine tracheal epithelia by using the hillocks and Ussing techniques. No significant baseline gland fluid flux ( J G) was measured by the hillocks technique after 3 min, and the epithelia had an average potential difference of 7.5 ± 0.5 mV (lumen negative) with a short-circuit current of 73 ± 4 μA/cm2, as measured by the Ussing technique. The secretagogue methacholine induced concentration-dependent increases in J G after 3 min from 0.003 μl · min−1 · cm−2 at 0.1 μM to 0.41 ± 0.04 μl · min−1 · cm−2 at 1,000 μM, with a 0.9 ± 0.1 mV hyperpolarization of the epithelium at 1,000 μM. When the epithelium was pretreated for 3 min with the sodium channel blocker amiloride, the methacholine (1,000 μM)-induced J G increased to 0.67 ± 0.09 μl · min−1 · cm−2, and the hyperpolarization increased to 2.2 ± 0.5 mV over the amiloride-pretreated level. When pretreated for 3 min with the chloride channel blocker diphenylamine-2-carboxylic acid, the methacholine (1,000 μM)-induced J G was inhibited to 0.20 ± 0.06 μl · min−1 · cm−2, and the methacholine-induced hyperpolarization was abolished. These data indicate that, in porcine airways, methacholine-induced J G may be increased by inhibition of sodium absorption and decreased by inhibition of chloride secretion.

We describe a computer-based image acquisition and analysis system designed for the quantitation of airway submucosal gland fluid flux by the hillocks technique. This technique is based on the detection of the increase in surface area of hillocks formed above gland duct openings by secretions captured under a thin layer of tantalum powder. The advantages of the system are: (a) ability to detect and measure individual submucosal gland secretions, (b) computer assisted data acquisition and analysis, and (c) decreased propagated error in hillock volume measurements. Test results of a practical implementation of the system demonstrate a swine tracheal submucosal gland flux induced by the muscarinic agonist acetylcholine (50 M) of 0.65 l/min/cm 2 . © 2001 Elsevier Science Ireland Ltd. All

We hypothesized that inhibition and activation of basolateral to luminal chloride transport mechanisms were associated with respective decreases and increases in basolateral to luminal water fluxes. The luminal to basolateral (J W L→B ) and basolateral to luminal (J W B→L ) water fluxes across ovine tracheal epithelia were measured simultaneously. The mean J W L→B (6.5 μl/min/cm2) was larger than J W B→L (6.1 μl/min/cm2). Furosemide reduced J W B→L from 6.0 to 5.6 μl/min/cm2. Diphenylamine-2-carboxylate (DPC) reduced J W B→L from 7.9 to 7.3 μl/min/cm2 and reduced the membrane potential difference by 38%. Furosemide together with DPC decreased J W L→B by 30% and J W B→L by 15%. Norepinephrine increased J W B→L from 4.9 to 6.0 μl/min/cm2. Neuropeptide Y in the presence of norepinephrine decreased J W L→B (6.4 to 5.2 μl/min/cm2) and returned J W B→L to its baseline value. Vasopressin increased J W B→L from 4.1 to 5.1 μl/min/cm2. Endothelin-1 induced a simultaneous increase in J W B→L (7.0 to 7.7 μl/min/cm2) and decrease in J W L→B (7.4 to 6.4 μl/min/cm2); and decreased the membrane resistance. These data indicate that in tracheal epithelia under homeostatic conditions J W B→L has a ∼15% actively coupled component. Consistent with our hypothesis, inhibition and receptor-induced stimulation of chloride effluxes were associated with decreases and increases in J W B→L , respectively. However, as inhibition of transcellular chloride transport always decreased J W L→B more than J W B→L , reducing transepithelial chloride transport did not result in less water being transported into the airway lumen.

In the search for the mechanisms whereby water is transported across biological membranes, we hypothesized that in the airways, the hydration of the periciliary fluid layer is regulated by luminal-to-basolateral water transport coupled to active transepithelial sodium transport. The luminal-to-basolateral (JWL-->B) and the basolateral-to-luminal (JWB-->L) transepithelial water fluxes across ovine tracheal epithelia were measured simultaneously. The JWL-->B (6.1 microliter/min/cm2) was larger than JWB-->L (4.5 microliter/min/cm2, p < 0.05, n = 30). The corresponding water diffusional permeabilities were PdL-->B = 1.0 x 10(-4) cm/s and PdB-->L = 7.5 x 10(-5) cm/s. The activation energy (Ea) of JWL-->B (11.6 kcal/mol) was larger than the Ea of JWB-->L (6.5 kcal/mol, p < 0.05, n = 5). Acetylstrophanthidin (100 microM basolateral) reduced JWL-->B from 6.1 to 4.4 microliter/min/cm2 (p < 0. 05, n = 5) and abolished the PD. Amiloride (10 microM luminal) reduced JWL-->B from 5.7 to 3.7 microliter/min/cm2 (p < 0.05, n = 5) and reduced PD by 44%. Neither of these agents significantly changed JWB-->L. These data indicate that in tracheal epithelia under homeostatic conditions, JWB-->L was dominated by diffusion (Ea = 4.6 kcal/mol), whereas approximately 30% of JWL-->B was coupled to the active Na+,K+-ATPase pump (Ea = 27 kcal/mol).