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R A Star

Publications and source records attributed to R A Star.

At least 19 recordsLinked to original sources

Imprecision of the hemodialysis dose when measured directly from urea removal. Hemodialysis Study Group.

BACKGROUND: The postdialysis blood urea nitrogen (BUN; Ct) is a pivotal parameter for assessing hemodialysis adequacy by conventional blood-side methods, but Ct is relatively unstable because of hemodialysis-induced disequilibrium. The uncertainty associated with this method is potentially reduced or eliminated by measuring urea removed on the dialysate side, a more direct approach that can determine adequacy from the fraction of urea removed and by substituting an estimate of the equilibrated postdialysis BUN (Ceq) for Ct. For a patient with a known urea volume (V), Ceq, the equilibrated Kt/V (eKt/V), and the solute removal index (SRI) can be calculated from the predialysis BUN (C0), total urea nitrogen removed (A), and V from simple mass balance calculations (dialysate/volume method). However, a theoretical error analysis showed that relatively small errors in A, C0, or V are magnified when SRI or eKt/V is calculated using this method, especially at higher eKt/V values (for example, if eKt/V = 1.4 per dialysis, a 7% dialysate collection error causes a 20% error in eKt/V). METHODS: During three to four baseline dialyses in each of 39 patients enrolled in the pilot phase of the HEMO Study, "A" was measured using an instrument that sampled dialysate frequently (Biostat), and V was calculated from A, C0, and Ceq (median CV for V = 5.6%). The mean V was then applied to the dialysate/volume method to estimate eKt/V and SRI during two to five subsequent dialyses per patient (comparison dialyses). The accuracy and precision of these estimates were assessed by comparing them with eKt/V and SRI derived from a direct measurement of Ceq drawn 30 minutes after dialysis (reference method), from mathematical curve-fitting of sequential dialysate urea concentrations (dialysate curve-fit method), and from another blood-side method that estimates eKt/V from single pool Kt/V and the fractional rate of solute removal (rate method): eKt/V = spKt/V - 0.6.K/V + 0.03. RESULTS: During 128 comparison dialyses, median absolute errors for calculated eKt/V compared with the reference method were 0.169, 0.061, and 0.071 for the dialysate/volume method, the rate method, and the dialysate curve-fitting method, respectively. The corresponding correlation coefficients were 0.47, 0.88, and 0.81. For SRI, median absolute errors were 0.044, 0.018, and 0.027, and the correlation coefficients were 0.54, 0.85, and 0.74 for the three methods. CONCLUSIONS: The precision of eKt/V and SRI measurements was significantly lower for the dialysate/volume method compared with the blood-side methods. Inclusion of the dialysate curve analysis provided by the Biostat restored precision to the dialysate method to a level comparable to that of the blood-side methods. New techniques employing dialysate urea analysis should include a concentration profile to avoid these inherent methodological errors and assure the accuracy of eKt/V and SRI.

Female

Alpha-melanocyte-stimulating hormone inhibits renal injury in the absence of neutrophils.

BACKGROUND: We previously showed that alpha-melanocyte stimulating hormone (alpha-MSH) decreases ischemia/reperfusion injury even when started six hours after ischemia. Alpha-MSH inhibits both neutrophil accumulation and nitric oxide production. To determine the relative importance of alpha-MSH on the neutrophil pathway, we examined the effects of alpha-MSH in injury models where neutrophil effects are minimal or absent. METHODS: We studied the effects of alpha-MSH in (1) intercellular adhesion molecule-1 (ICAM-1) knock-out and background mice that were subjected to 40 minutes of ischemia and 24 hours reperfusion, and (2) isolated kidneys that were subjected to in vivo ischemia for 20 minutes and then perfused ex vivo for one hour without neutrophils. To begin to search for direct tubule effects of alpha-MSH, we studied the effect of alpha-MSH on nitric oxide (NO) in endotoxin/interferon-gamma-treated mouse cortical tubule cells. RESULTS: ICAM-1 knock-out mice had 75% less neutrophil infiltration than background mice after ischemia. Despite the relative lack of neutrophils, alpha-MSH inhibited renal injury in ICAM-1 knock-out mice. Alpha-MSH also significantly preserved GFR and tubular sodium reabsorption in the isolated perfused ischemic kidney model. Alpha-MSH and a nitric oxide inhibitor did not exhibit synergy. Finally, alpha-MSH inhibited nitrite production by 20% in the mouse cortical tubule cells (MCT), similar to parallel observations in a cultured mouse macrophage line (RAW cells). CONCLUSIONS: We conclude that alpha-MSH decreases renal injury when neutrophil effects are minimal or absent, indicating that alpha-MSH inhibits neutrophil-independent pathways of renal injury. The preservation of sodium absorption ex vivo and inhibition of nitrite production in cultured MCT cells suggests that alpha-MSH inhibits tubular injury by direct tubular effects.

Animals

Localization of the ROMK potassium channel to the apical membrane of distal nephron in rat kidney.

BACKGROUND: The apical potassium (K+) channels mediate K+ recycling in thick ascending limb (TAL) and K+ secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K+ channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K+ channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. METHODS: Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. RESULTS: The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a approximately 42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H+-ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. CONCLUSIONS: These results further strengthen the idea that ROMK channels play important roles in the recycling of K+ in TAL and the secretion of K+ in CCD.

Animals

Treatment of acute renal failure.

Acute renal failure is a life threatening illness whose mortality has remained high since the introduction of hemodialysis 25 years ago, despite advances in supportive care. Acute renal failure is an extremely morbid and costly disorder with a significant proportion of patients progressing to end-stage renal disease requiring dialysis. To the nephrologist, acute renal failure remains an extremely frustrating disease, because the pathophysiology is not well understood and the limited therapeutic options force the nephrologist to sit on the sidelines and wait for renal function to return. For example, dialysis remains the only FDA-approved treatment for acute renal failure, but dialysis may also cause renal injury that prolongs renal failure. The purpose of this perspective is to understand the results of the recent, largely negative, clinical trials in view of recent advances in the epidemiology of ARF. This review will also discuss diagnostic tools, strategies for improved design of clinical trials, and other therapeutic interventions that will be needed to properly treat acute renal failure in the 21st century.

Acute Kidney Injury

alpha-Melanocyte-stimulating hormone and acute renal failure.

alpha-Melanocyte-stimulating hormone (MSH) is an endogenous anti-inflammatory cytokine that inhibits all major forms of inflammation, alpha-MSH level is increased at sites of inflammation in humans, and is produced in the pituitary and in macrophages. The effects of alpha-MSH are mediated by melanocortin receptors found on macrophages, neutrophils, and renal tubules. alpha-MSH inhibited ischemic acute renal failure in mice and rats, even when started 6 h after injury. alpha-MSH acts, in part, by inhibiting the maladaptive activation of genes that cause inflammatory and cytotoxic renal injury. However, alpha-MSH is effective even in the absence of neutrophils, suggesting that alpha-MSH also acts directly on renal tubules.

Acute Kidney Injury

Alpha-melanocyte-stimulating hormone protects against renal injury after ischemia in mice and rats.

Reperfusion after ischemia induces cytokines, chemoattractant chemokines, adhesion molecules, and nitric oxide (NO). The resultant neutrophil adherence and NO potentiates renal injury. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent anti-inflammatory agent that inhibits neutrophil migration and production of neutrophil chemokines and NO. Since neutrophils and NO promote renal ischemic injury, we sought to determine if alpha-MSH inhibits renal injury in a model of bilateral renal ischemia. alpha-MSH significantly reduced ischemia-induced renal damage, measured by changes in renal histology and plasma blood urea nitrogen and creatinine in mice. alpha-MSH significantly decreased tubule necrosis, neutrophil plugging, and capillary congestion. Delay of alpha-MSH treatment for 6 h after ischemia also significantly inhibited renal damage. alpha-MSH also significantly inhibited ischemic damage in rats. To begin to determine the mechanism of action of alpha-MSH, we measured its effects on mediators of neutrophil trafficking and induction of the inducible isoform of NO synthase-II. alpha-MSH inhibited ischemia-induced increases in mRNA for the murine neutrophil chemokine KC/IL-8. alpha-MSH also inhibited induction of mRNA for the adhesion molecule ICAM-1, which is known to be critical in renal ischemic injury. alpha-MSH inhibited nitration of kidney proteins and induction of NO synthase-II. We conclude: (a) alpha-MSH protects against renal ischemia/reperfusion injury; and (b) it may act, in part, by inhibiting the maladaptive activation of genes that cause neutrophil activation and adhesion, and induction of NO synthase.

Animals

Intrarenal localization of nitric oxide synthase isoforms and soluble guanylyl cyclase.

1. Nitric oxide (NO) plays an important role in the regulation of renal function. To date, five isoforms of NO synthase (NOS) and four subunits of soluble guanylyl cyclase have been cloned. The kidney contains four isoforms of NOS and all subunits of soluble guanylyl cyclase. 2. This review focuses on the intrarenal location of the isoforms of NOS and the subunits of soluble guanylyl cyclase.

Animals

Alpha-melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation.

Alpha-Melanocyte-stimulating hormone (MSH) is a potent anti-inflammatory agent in many models of inflammation, suggesting that it inhibits a critical step common to different forms of inflammation. We showed previously that alpha-MSH inhibits nitric oxide (NO) production in cultured macro-phages. To determine how alpha-MSH acts in vivo, we induced acute hepatic inflammation by administering endotoxin (LPS) to mice pretreated with Corynebacterium parvum, alpha-MSH prevented liver inflammation even when given 30 min after LPS administration. To determine the mechanisms of action of alpha-MSH, we tested its influence on NO, infiltrating inflammatory cells, cytokines, and chemokines. Alpha-MSH inhibited systemic NO production, hepatic neutrophil infiltration, and increased hepatic mRNA abundance for TNF alpha, and the neutrophil and monocyte chemokines (KC/IL-8 and MCP-1). We conclude that alpha-MSH prevents LPS-induced hepatic inflammation by inhibiting production of chemoattractant chemokines which then modulate infiltration of inflammatory cells. Thus, alpha-MSH has an effect very early in the inflammatory cascade.

Animals

Induction of inducible nitric-oxide synthase by the heterotrimeric G protein Galpha13.

While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.

Animals

alpha-MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line.

alpha-Melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide derived from pro-opiomelanocortin, has potent antiinflammatory activity in laboratory animals. alpha-MSH inhibits nitric oxide production by murine macrophages, an influence believed to reflect activation of an autocrine circuit in these cells, one that is based on production and release of alpha-MSH and subsequent stimulation of melanocortin receptors. We found that THP-1 cells, human monocytic cells, produced alpha-MSH; this production was increased by interleukin-6, tumor necrosis factor a, or concanavalin A. These cells also expressed the gene for the human alpha-MSH receptor MC1. Unlike murine macrophages, THP-1 cells produced little nitrite in response to interferon-gamma (IFN-gamma) and lipopolysaccharide, and a-MSH inhibited this production only slightly. However, production of neopterin, a presumed primate homologue of nitric oxide in lower animals, was increased in THP-1 cells stimulated with INF-gamma plus TNF-alpha and alpha-MSH significantly inhibited this production. The evidence indicates that an autocrine regulatory circuit based on alpha-MSH occurs in human monocyte/macrophages much as in murine macrophages. alpha-MSH-induced modulation of specific inflammatory mediators/cytotoxic agents appears to differ depending on the importance of the mediators in the myelomonocytic cells of different species.

Amino Acid Sequence

The neuropeptide alpha-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro.

The proopiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) has potent anti-inflammatory effects in all animal models of inflammation against which it has been tested. Understanding of the mechanism by which this occurs is incomplete, although there is recent evidence for alpha-MSH receptors in murine and human macrophages and for modulation of production of proinflammatory cytokines and related mediators by alpha-MSH. Because of the prominence of neutrophils in early stages of inflammatory reactions where alpha-MSH is effective, we examined human neutrophils for evidence of mRNA for alpha-MSH receptors and for inhibition of neutrophil chemotaxis. There was accumulation of mRNA for melanocortin receptor 1 (MC1) in RT/PCR product from neutrophils stimulated with interferon and LPS. In subsequent studies alpha-MSH inhibited migration of neutrophils from most normal volunteers when the cells were placed in FMLP or IL-8 gradients. The inhibition by alpha-MSH could be traced to alterations in cAMP in neutrophils. The presence of alpha-MSH receptor message in neutrophils is consistent with the established anti-inflammatory effects of the peptide. Direct inhibition of neutrophil chemotaxis likely contributes to the anti-inflammatory activity of alpha-MSH.

Analysis of Variance

Differential regulation of rat glomerular and proximal tubular renin mRNA following uninephrectomy.

Angiotensin II is thought to play a role in the renal adaptations to reduced renal mass, but earlier work has shown that plasma renin activity (PRA) does not increase in this setting. To examine this paradox, we studied the effect of uninephrectomy (UNX) on circulating, juxtaglomerular, glomerular, and proximal tubular (PT) renin. PRA was unchanged 2 wk following UNX and fell slightly at 6 wk. Single kidney renin secretory capacity and cortical renin mRNA, reflecting juxtaglomerular renin, were unchanged at 2 and 6 wk. With quantitative competitive reverse transcription-polymerase chain reaction, renin mRNA in microdissected glomeruli and PT were dramatically increased 2 wk post-UNIX (for glomeruli: sham, 1.2 +/- 0.3, vs. UNX, 8.8 +/- 1.9 x 10(5) copies/glomerulus; for PT: sham, 4.6 +/- 0.9, vs. UNX, 17.7 +/- 5.1 x 10(3) copies/mm). By 6 wk, glomerular renin was unchanged, and PT renin mRNA was markedly suppressed (for glomeruli; sham, 2.9 +/- 1.2, vs. UNX, 4.2 +/- 1.1 x 10(5) copies/glomerulus; for PT: sham, 7.5 +/- 2.1, vs. UNX, 1.0 +/- 0.3 x 10(3) copies/mm). These results demonstrate differential regulation of the circulating, juxtaglomerular, glomerular, and PT renin systems. Early activation of glomerular and PT renin may result in increased local generation of angiotensin II and thereby affect renal structural and functional adaptations following UNX.

Animals

Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of alpha-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and alpha-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon gamma. alpha-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. alpha-MSH increased cAMP accumulation in RAW cells, characteristic of alpha-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary alpha-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecular of alpha-MSH, was expressed in RAW cells, and tumor necrosis factor alpha increased production and release of alpha-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor alpha can induce macrophages to increase production of alpha-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that alpha-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines.

Amino Acid Oxidoreductases

Accuracy of urea removal estimated by kinetic models.

The most accurate method for assessing the dialysis dose delivered during high efficiency/flux hemodialysis has not been established. Most current indices of dialysis dose are based on blood-side urea measurements, and thus estimate urea removal. Unfortunately, these methods may lead to inappropriately short dialysis during high flux or high efficiency dialysis, perhaps because of inaccuracies in estimating the amount of urea removal. It is unknown whether these clearance-based approaches can accurately predict either absolute or fractional net urea removal, the latter being equivalent to the solute removal index (SRI). Therefore, we compared the urea removal calculated by five blood-side kinetic methods: (1) urea reduction ration, (2) 1-pool, (3) 2-pool models, and the (4) Smye and (5) Daugirdas formulae. These were compared with the gold standard measurement by direct dialysate quantification. Eight stable patients receiving high-flux hemodialysis were studied over four sessions each. BUN was measured at 0, 45 minutes, 90 minutes, end dialysis, one hour after dialysis (equilibrium value), and 48 hours later. Total body water was determined from the dialysate urea removal; the urea generation rate was calculated using one hour post-dialysis and 48-hour BUN values. Both the total body water and urea generation rate were provided to the 1- and 2-pool models to optimize accuracy. The urea reduction ratio overestimated SRI. The 1-pool model overestimated both absolute urea removal and SRI in 28 of 32 sessions. The 2-pool model slightly underestimated both absolute urea removal and SRI. In contrast, the Smye and Daugirdas formulas accurately estimated SRI.(ABSTRACT TRUNCATED AT 250 WORDS)

Female

TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells.

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.

Amino Acid Oxidoreductases

Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.

Prolonged hypoxia in the adult rat causes a decline in endothelium-derived nitric oxide (NO) production in the pulmonary circulation. To evaluate whether this is related to a decrease in endothelial NO synthase (NOS-III) expression, we determined the effects of hypobaric hypoxia (7 or 21 days) on NOS-III gene expression in adult rat lung. Neuronal NOS (NOS-I) expression was also examined; NOS-I has been immunohistochemically localized to rat bronchiolar epithelium. NOS-III and NOS-I mRNA abundance were assessed in reverse transcription-polymerase chain reaction assays and the proteins were evaluated by immunoblot analysis. After 7 and 21 days of hypoxia, there were increases in the steady-state levels of both NOS-III and NOS-I mRNA, rising 2.7- to 3.0-fold and 2.5- to 2.8-fold, respectively. These findings were confirmed by Northern analyses. In parallel, NOS-III and NOS-I protein abundance were also increased with hypoxia by 3.0- to 3.5-fold and 2.4- to 3.0-fold, respectively. NOS activity detected by [3H]arginine to [3H]citrulline conversion rose 109%. Thus, prolonged in vivo hypoxia causes enhancement of NOS-III and NOS-I gene expression in adult rat lung, indicating that the pulmonary expression of these genes is modulated in vivo. The increase in NOS-III expression does not explain the declines in pulmonary endothelial NO production previously observed following prolonged hypoxia in this model. Alternatively, the fall in NO production may be related to diminished NOS co-factor availability.

Amino Acid Oxidoreductases

Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx.

The mechanism of activation of the agonist-stimulated Ca2+ entry pathway in the plasma membrane is not known. To determine the role of nitric-oxide synthase (NOS) and cGMP in the regulation of this pathway, we used intact and streptolysin O (SLO)-permeable pancreatic acini and measured the relationship between Ca2+ release from internal stores, the NO metabolic pathway, generation of cGMP, and activation of Ca2+ entry. We found that agonist- or thapsigargin (Tg)-activated Ca2+ entry is inhibited by L-NA, a specific inhibitor of NOS, and by LY83583, an inhibitor of guanylyl cyclase. Inhibition of Ca2+ entry by inhibition of NOS was reversed by the NO releasing molecules NO2- and sodium nitroprusside (SNP) and by Bt2cGMP. Inhibition of Ca2+ entry by inhibition of guanylyl cyclase was reversed by Bt2cGMP, but not by the NO releasing agents. The use of L-NA-treated cells and different concentrations of SNP revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP up to 10-fold above control activated Ca2+ entry. Further increase in cGMP up to 80-fold above control inhibited Ca2+ entry in a concentration-dependent manner. Measurement of cellular cGMP in intact cells showed that carbachol, Tg, and NO2- increased cGMP to similar levels. The effects of carbachol and Tg were inhibited by L-NA and LY83586, whereas the effect of NO2- was inhibited only by LY83583. SLO-permeabilized cells were shown to be agonist-competent in that the agonist induced Ca2+ release from the inositol 1,4,5-trisphosphate (IP3) pool and activated a NO-dependent generation of cGMP. These cells were used to study the regulation of NOS by Ca2+ and by Ca2+ content of the internal stores. When internal stores were maintained loaded with Ca2+, increasing medium [Ca2+] up to 2.5 microM only modestly increased NOS activity. In contrast, the depletion of Ca2+ from internal stores markedly increased NOS activity independent of medium [Ca2+]. Thus, NOS senses both cytosolic [Ca2+]i and internal store Ca2+ load. We propose that activation of Ca2+ entry involves an agonist-mediated Ca2+ release from internal stores which activates a cellular pool of NOS to generate cGMP, which then modulates Ca2+ entry pathway in the plasma membrane. This mechanism can explain the capacitative nature of Ca2+ entry. The biphasic effect of cGMP provides the cells with a negative feedback mechanism which inhibits Ca2+ entry during periods of high cell [Ca2+]i. This could allow oscillatory behavior of Ca2+ entry.

Amino Acid Oxidoreductases

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct.

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl-/HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)-dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene-2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals