Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors.

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate I(Na)(sc) and I(Cl)(sc). By RT-PCR, two P2X receptor channels (P2X(3), P2X(4)) and two P2Y G protein-coupled receptors (P2Y(1), P2Y(2)) were identified. Functional localization of P2 purinoceptors suggest that I(Cl)(sc) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas I(Na)(sc) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit I(Na)(sc) across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate I(Cl)(sc) through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

Surgical management of the tight lip syndrome in the Shar-Pei dog.

The lower lip in some Shar-Pei puppies curls up over the mandible, causing lingual displacement of the incisor and canine teeth and may result in interference with the natural growth of the mandible. Surgical correction of this defect at the proper age can allow the mandible to grow normally and avoid development of an uncomfortable mouth. Two methods of correction are described. Cheiloplasty has been used by the author on 99 puppies over a 10 year period, only two of which were returned for additional surgery because the result was inadequate; the cheiloplasty procedure was repeated in these two dogs. Excision of an elliptical section of the skin of the chin was performed once during the same 10 year period.

Hyperosmolality inhibits sodium absorption and chloride secretion in mIMCD-K2 cells.

Previously we demonstrated that a cell line derived from mouse inner medullary collecting duct (mIMCD-K2) absorbs Na+ and secretes Cl- by electrogenic mechanisms and that arginine vasopressin (AVP) stimulates Cl- secretion. The objective of the present study was to determine whether hyperosmolality, both acute (minutes) and chronic (weeks), affects electrogenic Na+ absorption IscNa and electrogenic Cl- secretion IscCl across the IMCD. To this end, we measured IscNa and IscCl across monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. Osmolality was increased from 290 to 590 mosmol/kgH2O by adding 200 mosmol/kgH2O of NaCl and 100 mosmol/kgH2O of urea or 300 mosmol/kgH2O of sucrose to the bathing solutions. Acute and chronic hyperosmolality reduced basal IscNa and IscCl and the AVP-stimulated rise in IscCl. These findings indicate that osmolality is an important determinant of IscNa and IscCl across IMCD cells and that the osmolality of the interstitial fluid should be considered when evaluating the effects of hormones and other factors on Na+ and Cl- transport by the IMCD.

Detection of pathogen-related oral spirochetes, Treponema denticola, and Treponema socranskii in dental plaque from dogs.

Spirochetes have been observed in dental plaque from dogs, but specific spirochetes have not been identified. In particular, it is not known whether treponemes associated with periodontal diseases in humans also occur in dogs, and whether, like in humans, detection of specific treponemes correlates with periodontal status of dogs. Forty-two dogs were grouped according to the worst periodontal condition in the mouth, as determined by overt signs of inflammation and pocket probing depths. A representative specimen of dental plaque was obtained by pooling subgingival plaque collected from three uniform reference sites, irrespective of periodontal status at selected sites. The presence of pathogen-related oral spirochetes. Treponema denticola, and T. socranskii was determined using specific monoclonal antibodies in an immunocytochemical microscopic assay. All three treponemes were detected in all groups, but a significantly greater proportion of dogs with pocket probing depths > or = 5 mm had detectable treponemes, compared to dogs that were in periodontal health.

Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney.

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

Adenosine inhibits arginine vasopressin-stimulated chloride secretion in a mouse IMCD cell line (mIMCD-K2).

Previously, we demonstrated that a mouse inner medullary collecting duct cell line (mIMCD-K2) secretes Cl- by an electrogenic mechanism via cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995; D. Vandorpe, N. Kizer, F. Ciampolillo-Bates, B. Moyer, K. Karlson, W. B. Guggino, and B. A. Stanton. Am. J. Physiol. 269 (Cell Physiol. 38): C683-C689, 1995]. The objective of the present study was to determine whether adenosine, and adenosine A1 receptors (A1AR) specifically, regulate electrogenic Cl- secretion (IscCl) in mIMCD-K2 cells. Neither N6-cyclohexyladenosine (CHA), a specific A1AR agonist, nor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1AR antagonist, altered basal, unstimulated IscCl in monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. In contrast, DPCPX increased arginine vasopressin (AVP)-stimulated IscCl, an effect that was reversed by CHA. Adenosine deaminase (ADA), which oxidatively deaminates adenosine to inosine, increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of ADA on AVP-stimulated IscCl. These results suggest that adenosine, via A1AR, inhibits AVP-stimulated IscCl. To identify the source(s) of extracellular adenosine, we examined the effects of dipyridamole, an inhibitor of nucleoside transport, and alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP), an inhibitor of ecto-5'-nucleotidase, on AVP-stimulated IscCl. Both compounds increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of dipyridamole and AOPCP on IscCl. Neither ADA nor CHA had an effect on 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated IscCl. Moreover, U-73122, an inhibitor of phospholipase C, failed to attenuate the increase in AVP-stimulated IscCl elicited by dipyridamole and AOPCP or the decrease in AVP-stimulated IscCl elicited by CHA. We conclude that adenosine, released by a nucleoside transporter and formed extracellularly by the breakdown of AMP, binds to A1AR, and decreases AVP-stimulated IscCl in mIMCD-K2 cells by reducing intracellular cAMP levels.

The renal cGMP-gated cation channel: its molecular structure and physiological role.

Cyclic nucleotide-gated cation channels, which are permeable to monovalent and divalent cations, are expressed in a number of tissues. cDNAs encoding cGMP-gated cation channel subunits have been cloned in retinal rods, cones, olfactory neuroepithelium, pineal gland, aorta, testis, heart, and most recently kidney. Patch clamp studies have identified and characterized cGMP-gated cation channels in the cortical collecting duct (CCD) and inner medullary collecting duct (IMCD). cGMP-gated cation channels in kidney share many biophysical and molecular properties with the retinal rod cGMP-gated channel. However, unlike the retinal rod channel, the cGMP-gated cation channel in kidney is inhibited by cGMP and stimulated by increased calcium levels. In the IMCD the cGMP-gated cation channel mediates electrogenic sodium absorption which is inhibited by ANP via cGMP. Recently, cGMP-gated cation channel poly(A+) RNA has been identified in other nephron segments by RT-PCR and in situ PCR hybridization. Furthermore, cGMP-gated cation channel protein has also been identified in all nephron segments by Western blot analysis. These observations suggest that cGMP-gated cation channels, or closely related gene products, may play an important physiological role in all nephron segments. Hormones that increase intracellular cGMP may regulate sodium, and perhaps calcium, uptake in nephron segments proximal to the IMCD. Increases in cell sodium and calcium may regulate other transport and signaling pathways.

Swim training alters sympathoadrenal and endocrine responses to hemorrhage in borderline hypertensive rats.

Swim training alters cardiovascular, sympathoadrenal, and endocrine responses to hemorrhage in borderline hypertensive rats (BHR). The effects of 10, 20, and 30% blood volume hemorrhages on cardiovascular, sympathoadrenal, and endocrine function in swim-trained (T; 2 h/day, 5 day/wk for 10-12 wk) and age-matched, untrained, sedentary, control (UT) borderline hypertensive rats (BHR) were assessed. Heart rate (HR) in UT BHR was significantly greater during the baseline (rest) period than T BHR. HR increased slightly from baseline in both groups after 10% hemorrhage but was significantly decreased in both groups after 20 and 30% hemorrhages. The decrease was eliminated by atropine (1 mg/kg iv). Systolic (SBP) and diastolic (DBP) blood pressures decreased significantly after 20 and 30% hemorrhages in both T and UT BHR but were not different between the groups at these times. Plasma norepinephrine levels were significantly increased above baseline after 20 and 30% hemorrhages in UT BHR and were significantly greater in UT BHR than T BHR after 30% hemorrhage. Plasma glucose levels increased significantly after 30% hemorrhage in both groups but were significantly greater in UT BHR than T BHR. Both plasma norepinephrine and plasma epinephrine levels showed strong positive correlations with plasma glucose. After 20 and 30% hemorrhages, plasma insulin levels were unchanged in T BHR but were significantly decreased in UT BHR. Plasma insulin levels were significantly less in UT than T BHR after 30% hemorrhage. These results suggest that swim training alters the effect that hemorrhage exerts on endocrine and sympathoadrenal function in BHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells.

A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.

Cloning of a cGMP-gated cation channel from mouse kidney inner medullary collecting duct.

The cDNA sequence coding for the cGMP-gated cation channel expressed in the mouse kidney inner medullary collecting duct has been determined. The kidney cGMP-gated cation channel cDNA has an open reading frame of 2055 nucleotides and encodes a 685 amino acid protein. One cDNA clone is alternatively spliced thereby producing a deletion of 107 bp. Two differentially spliced 5' untranslated regions were determined by 5' RACE.

Swim training alters renal and cardiovascular responses to stress in borderline hypertensive rats.

The present study assessed the effects of 40 min of tail shock stress (1-s shock, 0.2 mA every 30 s) on renal and cardiovascular function in swim-trained (T), water-immersed (IM), and untrained (UT) borderline hypertensive rats (BHR). T BHR swam for 2 h/day 5 days/wk for 10-12 wk, whereas IM BHR on the same schedule were placed in water at neck level and were not permitted to swim. Age-matched sedentary controls were paired one each with the exercise group (group 1) and the immersion group (group 2). Heart rate was significantly greater in UT than in T BHR (P = 0.09) during baseline (rest). Heart rate responses during stress were not different between UT and IM BHR (group 2). Systolic and diastolic blood pressures during stress and recovery were not different between UT BHR and T or IM BHR. Urine flow rate was significantly increased from baseline during the first 20 min of stress in UT and IM BHR only. Changes in glomerular filtration rate were not consistent across studies. Renal blood flow decreased significantly from baseline during tail shock stress in UT but not T BHR. Plasma glucose levels were significantly increased above baseline during the second 20 min of stress in UT BHR only and were significantly greater than those in the T BHR. Plasma insulin levels in UT BHR were significantly decreased from baseline during tail shock stress and recovery but were unchanged from baseline in T BHR. These observations suggest that swim training independent of water immersion alters the effect that stress exerts on renal and cardiovascular function in BHR, which results in better fluid and electrolyte conservation in T BHR.

Functional localization of adenosine receptor-mediated pathways in the LLC-PK1 renal cell line.

The functional localization of three adenosine receptor-mediated signal transduction pathways in the LLC-PK1 renal cell line was investigated. LLC-PK1 cells were grown on Millicell-CM filter inserts, which allow for the independent exposure of the apical or basolateral side of a confluent cell monolayer to hormones. Adenosine stimulated inositol phosphate turnover, inhibition of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (A1 receptor), and stimulation of cAMP accumulation (A2 receptor). Adenosine (10 microM) selectively applied to the basolateral side induced a significant (P < 0.05) increase in inositol phosphates, whereas apical exposure did not. The adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (1 microM), blocked the stimulation of inositol phosphate production in LLC-PK1 cells, provided support for an adenosine receptor-mediated event. When adenosine (30 nM) was selectively applied to the apical side, forskolin-stimulated cAMP levels were not significantly decreased (approximately 8%, P > 0.05). However, adenosine (30 nM) presented to the basolateral side produced a significant decrease (approximately 23%, P < 0.05) in forskolin-stimulated cAMP levels. A high dose (100 microM) of adenosine elicited a significant increase (P < 0.05) in cAMP levels when presented to either the apical or the basolateral cell surface. Adenosine (100 microM) applied to the apical side elicited significantly higher cAMP levels (P < 0.05) than the same dose applied basolaterally. LLC-PK1 cells grown on permeable supports exhibit a polarity of functional responses following activation by adenosine. These data support a topographic separation of the multiple adenosine signaling systems in a renal epithelial cell line.

Transient and steady-state cardiopulmonary responses to combined rhythmic and isometric exercise.

The transient and steady-state cardiopulmonary responses to combined rhythmic (R) and isometric (I) exercise were examined in nine subjects. Isometric exercise at 30% maximal voluntary contraction (MVC) was started 1.5 min prior to either a 50% or 75% maximal oxygen uptake (VO2max) cycle ride and continued for 1.5 min into the 10-min R. Systolic (Pas) and diastolic (P(ad)) blood pressure, heart rate (fc), inspired ventilation volumes (VI), and oxygen uptake (VO2) were recorded every 30 s throughout each experiment. Responses to I effort alone were recorded for comparison with experiments in which the combined exercises were performed during the first 1.5 min when R had not yet begun. Pas responses in the first 1.5 min of I (no R) showed the typical rapid linear increase. Addition of the R effort further increased Pas to levels which remained nearly constant (steady state) throughout R. R alone produced a slower Pas increase to approximately the same steady-state levels as those of the combined R and I exercise. For P(ad), the linear increase which occurred during the first 1.5 min of I was attenuated with the superimposition of R. Following cessation of I, P(ad) fell rapidly during continued R to levels not different from experiments with R alone. The fc during I alone increased slightly. As I continued, the onset of the R induced a further rapid increase in fc to levels not different from R alone. The VI showed a similar response to fc. VO2 during I alone did not change significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiopulmonary responses to combined rhythmic and isometric exercise in humans.

A rhythmic (R) and an isometric (I) exercise were performed separately and in combination to assess their additive effects on arterial systolic (P(as)) and diastolic (P(ad)) blood pressures, heart rate (fc), and minute ventilation (VI). The isometric effort consisted of a 40% maximal voluntary handgrip contraction (MVC) performed for a duration of 80% of a previously determined 40% MVC fatiguing effort. The R effort consisted of a 13-min cycle effort at 75% maximum oxygen consumption (VO2max). For the combined efforts, I was performed starting simultaneously with or ending simultaneously with R. Data on nine subjects yield statistically significant evidence (P less than 0.05) that the effects of I and R are not additive for the following three cases: (1) P(as) when I and R are ended simultaneously (I alone = 4.9, SEM 0.5 kPa increase; R alone = no significant change from steady state; I + R = 1.2, SEM 0.4 kPa increase), (2) P(ad) when I and R are started simultaneously (I alone = 4.1, SEM 0.4 kPa increase; R alone = 0.7, SEM 0.3 kPa decrease; I + R = 1.9, SEM 0.4 kPa increase), and (3) P(ad) when I and R are ended simultaneously (I alone = 4.1, SEM 0.4 kPa increase; R alone = 0.3, SEM 0.5 kPa decrease; I + R = 0.8, SEM 0.3 kPa increase). For all other variables and cases, there is not sufficient evidence to conclude that the effects of I and R are not additive. We conclude that R and I exercises do not invariably produce strictly additive cardiopulmonary responses.(ABSTRACT TRUNCATED AT 250 WORDS)