Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels.

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.

Enantio- and diastereoselective reductive aldol reactions with iridium-pybox catalysts.

[see reaction]. A catalytic amount of [(cod)IrCl]2 and indane-pybox converts diethylmethylsilane, methyl acrylate, and certain aldehydes to the derived reductive aldol adduct with good enantio- and diastereocontrol.

Etiologic factors associated with anterior knee pain in distance runners.

PURPOSE: The objectives of this study were 1) to examine the differences between a noninjured (C) cohort of runners (N = 70) and runners afflicted with anterior knee pain (AKP) according to selected training, anthropometric, rearfoot motion, ground reaction force, and muscular strength and endurance variables; 2) to explore multivariate relationships among these measures in the well and injured groups; and 3) to develop specific hypotheses concerning risk factors for injury that will later be tested in a prospective clinical study. METHODS: High speed videography (200 frames x s(-1)), a force platform (500 Hz), and a Cybex II+ isokinetic dynamometer were used to assess rearfoot motion, ground reaction forces, and knee muscular strength and endurance, respectively. A linear discriminant function was performed on each of the five categories of variables and revealed 19 significant (P < or = 0.05) predictors. These variables were then combined and a final discriminant function analysis was performed. RESULTS: Pronation through the first 10% of stance, arch index, shoe mileage, and extension peak torque were the best overall (P < or = 0.05) predictors. The AKP group had smaller mean values on all four significant predictors. CONCLUSION: With the exception of shoe mileage, which is likely a response to rather than a risk factor for AKP, these results should prove useful to clinicians in identifying runners at risk for anterior knee pain.

Communication of research and practice knowledge in nursing literature.

BACKGROUND: Previous research did not indicate whether communication occurred between the research and practice components of nursing knowledge. OBJECTIVES: This study was designed to examine the extent of communication between or within the nursing-research and nursing-practice components of the nursing literature. METHODS: Specific citing/cited relationships from the Current Index to Nursing and Allied Health Literature (CINAHL) are analyzed using citation analysis. The sample was taken from the population of indexed documents that contained references, that had the subject code of nursing, and that were originally published in the United States or Canada in 1989. RESULTS: The results show that authors of research articles tend to cite research articles more often than practice articles, and that authors of practice articles tend to cite practice articles more often than research articles. Authors with research degrees publish research articles more often than would be expected by chance, and authors with clinical and undergraduate degrees publish practice articles more often than expected. Authors associated with research institutions tend to publish research articles, and those affiliated with service institutions tend to publish practice articles. Most cited documents (69.4%) are from disciplines other than nursing. CONCLUSIONS: The existence of communication between the research and the practice component of nursing knowledge was demonstrated and appears to be more prevalent than previously indicated. Further research on how the cited literature is used would enable us to know about the depth and quality of the communication event and to note variations as the body of nursing knowledge evolves.

An evidence-based project for evaluating strategies to improve knowledge acquisition and critical-thinking performance in nursing students.

This longitudinal, quasi-experimental study with 142 junior nursing students focused on measurement of learning outcomes in two areas: acquisition of knowledge and development of critical thinking skills. The variation in clinical teaching strategy (structured versus unstructured health pattern assessment) was the independent variable. Results indicated significant gains in both knowledge and critical thinking performance from the beginning to the end of the semester. The significant gains in critical thinking performance provides support to the assertion that domain-specific measures of critical thinking are needed in nursing education. Additionally, our results suggested that it was the interaction between learning strategy and the characteristics of the learner that was more significant in determining knowledge improvement than the particular strategy. As a result of this study, it is recommended that faculty develop and use an evidence-based model to support their decision making regarding teaching methodologies. This seems especially relevant for large, introductory clinical courses that use team teaching to achieve educational goals related to improvement in critical thinking or knowledge.

Effect of partial ureteral obstruction on results of renal scintigraphy in dogs.

OBJECTIVE: To use scintigraphy to determine the effects of partial ureteral obstruction on renal transit time and induction of diuresis in dogs. ANIMALS: 8 adult dogs. PROCEDURE: Scintigraphy was performed, using technetium Tc 99m diethylenetriaminepentacetic acid (Tc 99m-DTPA), before and within 2 weeks after surgical induction of unilateral partial ureteral obstruction. Time of peak (TOP) for the parenchyma (pTOP) and whole kidney (wTOP) and mean-transit time (MTT) for the parenchyma (pMTT) and whole kidney (wMTT) were determined by evaluation of renal time-activity curves before and after deconvolution analysis. Percentage uptake for each kidney between 1 and 3 minutes after injection of Tc 99m-DTPA was determined and used to indicate glomerular filtration rate. The effect of diuresis was determined by measuring the slope of decrease in activity after i.v. administration of furosemide. Obstruction was documented by direct inspection of the ureter. RESULTS: There was a concomitant increase in pTOP, wTOP, pMTT, and wMTT of the kidney with the partially obstructed ureter in all dogs at various times between 2 and 9 days after surgery. Concurrently, renal time-activity curves changed shape. Percentage renal uptake of the affected kidney was decreased in 2 dogs. Response to furosemide injection was inconsistent for kidneys before surgery and for kidneys with obstructed and nonobstructed ureters after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Scintigraphy may be a useful procedure for the evaluation of renal function in dogs with ureteral obstruction. Induction of diuresis appears to be of little value for differentiating renal function in dogs with obstructed and nonobstructed ureters.

Incomplete urethral duplication with cyst formation in a dog.

Incomplete urethral duplication with cyst formation was diagnosed in a dog that had soft, fluctuant, subcutaneous swellings in the ventral perineal and penile areas and a history of nocturia and incontinence during recumbency that were unresponsive to treatment with antibiotics. Retrograde urethrocystography, voiding urethrography, double-contrast cystography, radiography after direct administration of contrast medium into cystic structures, and excretory urography were performed to evaluate the urinary tract. Communication between the cysts and the urethra was demonstrated radiographically only after intralesional injection of contrast medium. Nocturia and incontinence resolved after surgical removal of the urethral duplication and cysts. The dog was clinically normal 1 year after surgery.

Acetylcholine-activated chloride current in the T-84 colonic cell line.

The stimulation of epithelial chloride secretion by hormones and neurotransmitters involves the activation of apical membrane chloride channels. The regulation of chloride current by acetylcholine in the T-84 colonic cell line was investigated using single-channel patch-clamp techniques. Treatment with carbachol resulted in the stimulation of transient chloride currents in 18 of 32 previously quiescent patches. Lack of resolvable single-channel openings suggests that single-channel conductance is less than 5-pS. Of 18 responsive patches, 4 showed multiple current oscillations. Treatment of the cells with AlF4- activated sustained chloride currents, suggesting that G proteins are involved. In excised patches, chloride current was markedly sensitive to free Ca2+ concentrations between 500 and 1000 nM. Time-dependent activation and inactivation of chloride current occurred at +60 and -60 mV. These results indicate that the chloride channels responsible for cholinergic activation of chloride conductance in the T-84 colonic cell line are members of the very low conductance family of chloride channels.

Downregulation of a human colonic sialyltransferase by a secondary bile acid and a phorbol ester.

Fecal constituents such as bile acids and increased sialylation of membrane glycoproteins by alpha-2,6-sialyltransferase (HST6N-1) may contribute to colorectal tumorigenesis. We hypothesized that bile acids and phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] would upregulate HST6N-1 in colonic cells. However, deoxycholate (DOC) (300 mumol/l), a secondary bile acid, and TPA (20 ng/ml) decreased expression of an approximately 100-kDa glycoprotein bearing alpha-2,6-linked sialic acid in a colon cancer cell line (T84) in vitro. HST6N-1 mRNA levels were reduced approximately 80% by treatment (< or = 24 h) with DOC or TPA but not by cholate, a primary bile acid. Treatment (24 h) with DOC or TPA decreased activity of this enzyme to 30% and 13% of control, respectively. These effects of DOC and TPA were transcriptional and were mediated by Ca2+ and protein kinase C, respectively. Thus DOC and TPA both downregulated, and did not upregulate, alpha-2,6-sialyltransferase expression in vitro, but by different transduction pathways. As colorectal tumors grow, their progressive removal from the fecal milieu that normally downregulates this enzyme may favor invasion and metastasis.

Pediatric nurses' use of behaviors to make medication administration decisions in infants recovering from surgery.

Although behavioral observation is recommended as the primary pain assessment for the nonverbal postsurgical child, little is known about clinicians' use of observation in their medication administration decisions. Eight infants were videotaped after surgery and segments of the videotapes were categorized as medication inactive or medication active (assumed to relieve pain) based on the usual duration of infants' analgesics. Nurses (N=50) viewed these segments and mean percent agreement with the pharmacologic categorization was 54%. Agreement was high for medication active segments and low for medication inactive ones. Nurses reported using the pain behaviors described in the literature as well as other infant characteristics in their decision making. Infant behaviors observed in the medication inactive snippets were not suggestive enough of the presence of pain to result in the nurses choosing to medicate.

G proteins activate ionic conductances at multiple sites in T84 cells.

We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K+ equilibrium potential (E(K)) or Cl- equilibrium potential (E(Cl)) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K+ current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K+ current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), to pipettes stimulated large oscillating K+ and inward currents. This occurred even when Ca2+ was absent from the bath but not when the Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K+ was replaced with Na+ and cells were voltage clamped between E(Na) and E(Cl), GTPgammaS activated oscillating Na+ and Cl- currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to pipettes activated large oscillating K+ currents but only small inward currents. These results suggest that a carbachol-induced release of Ca2+ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P3 signaling pathway. In addition, this or another G protein activates Cl- current by directly gating Cl- channels to increase their sensitivity to Ca2+.

The evolution of a profession: where do boards of nursing fit in?

Differentiation between the structure and functions of Boards of Nursing and the professional association is important to understanding the evolution of nursing as a profession. This descriptive exploratory study revealed that Boards of Nursing usually have 5 members who are appointed by and can be removed by the governor of the state. All Boards are responsible for education and licensing at the professional entry-level but may not be responsible beyond that level. In contrast to the professional association, Boards report appreciable variability in structure and administration. Boards differ from the professional association in source of power and focus on the protection of the public rather than improvement of practice.

Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84)

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

Transport of glucose polymer-derived glucose by rabbit jejunum.

Mechanisms for the assimilation of glucose polymers have been inferred from perfusion studies. To further define these mechanisms, the results of measurements of unidirectional glucose fluxes across short-circuited rabbit jejunal segments in vitro are reported. Glucose polymer-stimulated short-circuit current was similar to that of glucose [19 +/- 6.0 microA/cm2 (n = 7) and 26 +/- 5.7 microA/cm2 (n = 13), respectively] and was inhibited by both acarbose and phlorizin. Acarbose, an alpha-glucosidase inhibitor with no effects of glucose transport, was used to uncouple digestion from absorption. Mucosal-to-serosal flux of glucose polymer-derived glucose was lower than that of an equal weight/volume of glucose [124 +/- 62 nmol.h-1.cm-2 (n = 4) vs. 452 +/- 121 nmol.h-1.cm-2 (n = 6); P less than 0.05] and was inhibited by both phlorizin and acarbose. No glucose polymers were detected in the serosal bath solutions by thin-layer chromatography. It is concluded that glucose polymer-derived glucose is transported by a phlorizin-inhibitable process at a rate slower than that of free glucose, a finding that suggests that hydrolysis limits glucose polymer assimilation.

Cholinergic stimulation produces oscillations of cytosolic Ca2+ in a secretory epithelial cell line, T84.

The effects of carbamylcholine (carbachol) on intracellular Ca2+ concentration ([Ca2+]c) of T84 cells were examined using the fluorescent Ca2+ indicator fura-2 and microfluorometric techniques. In single isolated cells, carbachol (100 microM) caused a rapid increase in [Ca2+]c of 184 +/- 15 nM (SE, n = 44) from a resting value of 56 +/- 7 nM. This initial transient was followed by a series of oscillations in 68% of the cells. Atropine (10 microM) blocked this response. Removal of bath Ca2+ did not inhibit the rise in [Ca2+]c or oscillations, but the response duration was shortened in 47% of the cells. The amplitude and latency of the initial Ca2+ rise, frequency of oscillations, and number of responding cells varied with the agonist concentration. We have previously shown that carbachol induces an oscillating K+ conductance in T84 cells [D. Devor, S. Simasko, and M. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990]. Simultaneous measurement of membrane K+ current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca2+]c and increase in K+ current. These results show that a rise in [Ca2+]c and oscillations is likely to underlie the membrane K+ current responses to carbachol in T84 cells. Responses from a single cell within a subconfluent monolayer were different from those of isolated cells. In cells of a monolayer the initial [Ca2+]c rise (111 +/- 8 nM; n = 41) was followed by a decline to a stable plateau, and oscillations were not seen. Removal of bath Ca2+ both reduced the initial transient and eliminated the plateau phase of the response. These results suggest that cell-to-cell contact or differentiation during monolayer formation influences the Ca2+ handling mechanisms of T84 cells.

Interaction of heptanol and pressure on sodium and chloride transport by toad skin.

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.

Intracellular pH and membrane potassium conductance in rabbit distal colon.

Intracellular pH (pHc) was measured in the short-circuited epithelium of rabbit distal colon using H(+)-selective microelectrodes. pHc was 6.91 +/- 0.02 (SE) when the bath pH was 7.4. Intracellular HCO3- activity (acHCO3-) was estimated from these measurements to be 8 +/- 0 mM. When we replaced all Cl- in the tissue bathing solutions with the impermeant anion gluconate, pHc rose to 7.44 +/- 0.08 and acHCO3- increased to 30 +/- 6 mM. These results demonstrate that this tissue contains a Cl(-)-HCO3- exchange mechanism. During the Cl- replacement the apical membrane electrical potential difference hyperpolarized from -55 +/- 1 to -74 +/- 3 mV, suggesting that membrane ionic conductance had changed. Elevation of either the apical or basolateral membrane bathing solution K+ concentration produced a greater depolarization of membrane potential during Cl- replacement than when tissues were bathed in normal electrolyte solutions. In additional experiments, pHc was raised by lowering the bath CO2 concentration while the bath Cl- concentration was kept normal. Under these conditions, membrane potential hyperpolarized and was more sensitive to the elevation of bath K+ concentration than when pHc was normal. These results suggest that membrane K+ conductance in this tissue is increased by intracellular alkalinization.