Showing papers in "American Journal of Physiology in 1995"
TL;DR: Seven classic and recently proposed methods used for the estimation of total arterial compliance have been evaluated for their accuracy and applicability in different physiological conditions and it is shown that the methods based on the two-element windkessel (WK) model are more accurate than thosebased on the three-element WK model.
Abstract: Seven classic and recently proposed methods used for the estimation of total arterial compliance have been evaluated for their accuracy and applicability in different physiological conditions. The pressure and flow data are taken from a computer model that provides realistic simulations of the nonlinear-distributed systemic arterial tree. Besides the great flexibility in simulating different physiological or pathological cases, the major advantage of the computer model is that it allows precise knowledge of the pressure-dependent total arterial compliance, which is the variable of interest. The results show that the methods based on the two-element windkessel (WK) model are more accurate than those based on the three-element WK model. The classic exponential decay and the diastolic area method yield essentially similar results, and their compliance estimates are accurate within 10% except at high heart rates. The later part of diastole, i.e., from the time that the systolic pressure wave has reached all peripheral beds, gives the best results. The newly proposed two-area and pulse pressure methods, both based on the two-element WK model, are accurate (errors in general or = 25%). The errors in the methods based on the three-element WK model arise from the fact that the input impedance in that model deviates significantly from the true input impedance at low frequencies. The strong dependence of compliance on pressure (elastic nonlinearity) does not invalidate the compliance estimates.(ABSTRACT TRUNCATED AT 250 WORDS)
288 citations
TL;DR: In myocardium that showed improved function after revascularization, preoperative flow and glucose uptake were higher by PET than in persistently dysfunctionalmyocardium, whereas preoperative 18F-labeled deoxyglucose uptake correlated positively with the amount of cardiomyocytes showing excess glycogen stores.
Abstract: Assessment of regional myocardial perfusion and glucose uptake with positron emission tomography (PET) identifies dysfunctional myocardium that shows improvement in function after revascularization. Yet little is known about the ultrastructural patterns of ischemic injury in myocardium with and without postoperative functional improvement in relation to residual perfusion and metabolism. Therefore dynamic PET with [13N]ammonia and 18F-labeled deoxyglucose was performed in 24 patients with coronary artery disease and anterior wall dysfunction undergoing bypass surgery. Transmural biopsies were obtained from the dysfunctional area during surgery and analyzed by optical and electron microscopy to quantify the presence of fibrosis and cardiomyocytes. As judged from the postoperative changes in contraction, left ventricular function improved in 16 patients. In myocardium that showed improved function after revascularization, preoperative flow and glucose uptake were higher by PET than in persistently dysfunctional myocardium. In tissue samples from myocardium that improved postoperatively, there were more cardiomyocytes, including a larger proportion of cells with excess glycogen stores (35 vs. 21%), and there was less fibrosis (24 vs. 49%) than in tissue samples from myocardium that did not improve functionally. Preoperative perfusion and postoperative wall motion were inversely correlated with the amount of tissue fibrosis, whereas preoperative 18F-labeled deoxyglucose uptake correlated positively with the amount of cardiomyocytes showing excess glycogen stores.
196 citations
TL;DR: Data suggest a possible differential sympathetic neural control over catecholamine-induced lipolysis and that lipolytic rates are greater for internal vs. external WAT pads.
Abstract: When Siberian hamsters are transferred from long summerlike days (LDs) to short winterlike days (SDs) they decrease their body weight, primarily as body fat. These SD-induced decreases in lipid stores are not uniform. Internally located white adipose tissue (WAT) pads are depleted preferentially of lipid, whereas the more externally located subcutaneous WAT pads are relatively spared. These data suggest a possible differential sympathetic neural control over catecholamine-induced lipolysis and that lipolytic rates are greater for internal vs. external WAT pads. Moreover, if these differential rates of lipolysis are due to differential sympathetic nervous system (SNS) drives on the pads, then fat pad-specific catecholaminergic innervation may exist. Therefore, we tested whether inguinal WAT (IWAT; an external pad) and epididymal WAT (EWAT; an internal pad) were innervated differentially. In addition, we tested whether norepinephrine (NE) turnover (TO) reflected the presumed greater SNS drive on EWAT vs. IWAT after SD exposure. Injections of fluorescent tract tracers [Fluoro-Gold or indocarbocyanine perchlorate (DiI)] demonstrated projections from the SNS ganglia T13-L3 to both fat pads. Retrograde labeling revealed a relatively separate pattern of distribution of labeled neurons in the ganglia projecting to each pad. In vivo anterograde transport of DiI resulted in labeling in both IWAT and EWAT that included staining around individual adipocytes and occasionally retrogradely labeled cells. The proportionately greater decrease in EWAT compared with IWAT mass after 5 wk of SD exposure was reflected in greater EWAT NE TO than found in their LD counterparts for this pad.(ABSTRACT TRUNCATED AT 250 WORDS)
181 citations
TL;DR: Basal hypercortisolemia and, at baseline, heart rates were higher in depressed patients, regardless of age, and blood pressure was elevated in older healthy controls as well as depressed patients.
Abstract: Aging and hypercortisolism may be associated with alterations of stress-induced hormone release. We therefore studied 20 normal controls of two different age groups ( 60 yr of age) and 20 age-matched patients with major depression; baseline ACTH and cortisol secretion (between 1400 and 1700) as well as blood pressure and heart rate and their responses to a 45-min lasting signal detection task (1705-1750) were determined. No difference in basal hypothalamic-pituitary-adrenal (HPA) system activity between young and older healthy controls was noted. The cognitive challenge resulted in an increase in stress-induced hormonal secretion that was greater in the older controls than in their young counterparts. Basal hypercortisolemia and, at baseline, heart rates were higher in depressed patients, regardless of age. Blood pressure was elevated in older healthy controls as well as depressed patients. With the exception of the young depressed patients, all groups responded with an increase of the cardiovascular parameters during stress.
127 citations
TL;DR: It is concluded that in cultured rat ventricular myocytes, IL-1 beta suppresses ICa,L via a PTX-insensitive G protein through the signal transduction pathway of interleukin-1 Beta through the inhibition of acetylcholine on the L-type Ca2+ current.
Abstract: The effect and possible signal transduction pathway of interleukin-1 beta (IL-1 beta) on the L-type Ca2+ current (ICa,L) in cultured adult rat ventricular myocytes were examined using whole cell patch-clamp techniques. When myocytes were internally dialyzed with a solution containing GTP, IL-1 beta caused a concentration-dependent decrease in the peak ICa,L (Ba2+ as the charge carrier). IL-1 beta did not significantly alter the voltage dependence of the peak ICa,L nor the steady-state inactivation and activation, but did slightly slow the rate of inactivation. In myocytes dialyzed with solutions without GTP or including guanosine 5'-O-(2-thiodiphosphate) to replace GTP, IL-1 beta had no effect on ICa,L. In contrast, when guanosine 5'-O-(3-thiotriphosphate) was used to replace GTP, the suppression of ICa,L induced by IL-1 beta remained. Preincubation of myocytes with pertussis toxin (PTX), which completely abolished the acetylcholine effect on isoproterenol-stimulated ICa,L, had no effect on the inhibitory action of IL-1 beta on ICa,L. We conclude that in cultured rat ventricular myocytes, IL-1 beta suppresses ICa,L via a PTX-insensitive G protein.
117 citations
TL;DR: The results show that NOS activity and gene expression are inversely related to chronic changes in renal perfusion, salt balance, and salt transport at the distal tubule in parallel with the known response of renin to these changes.
Abstract: Four chronic experiments were performed to assess changes in the activity and gene expression of type I nitric oxide synthase (NOS) at the macula densa (MD) and of renin expression and immunoreactivity (IR) at the juxtaglomerular apparatus (JGA) of rat kidney, as follows: 1) two-kidney, one-clip Goldblatt hypertension (2K1C, for 3 and 40 days; sham operation for controls), 2) furosemide treatment (150 mg/kg-1.day-1 ip for 5 days), 3) chronic low-salt diet (0.02%) vs. high-salt diet (3%; both for 11 days), and 4) chronic blockade of NOS by nitro-L-arginine methyl ester (L-NAME, 40 mg.kg-1.day-1 for 2 mo). NOS and renin gene expression, NOS enzyme activity and renin IR were semiquantitatively evaluated with histochemical methods (NADPH diaphorase, in situ hybridization, immunohistochemistry). In 2K1C, marked increases were induced in NOS and renin in the ischemic vs. contralateral kidneys both after 3 and 40 days, respectively (P < 0.05). Related to controls, significant increases in the ischemic kidney were encountered after 3 and 40 days, whereas contralateral suppression of NOS and renin was found only after 40 days. Furosemide treatment resulted in a marked increase of both NOS and renin levels compared with controls (P < 0.05). Salt restriction induced a significant elevation of NOS levels compared with salt loading (P < 0.05), whereas only minor changes were evident in renin levels. L-NAME treatment resulted in a moderate reduction of NOS activity (not significant), whereas renin levels were markedly reduced (P < 0.05). These results show that NOS activity and gene expression are inversely related to chronic changes in renal perfusion, salt balance, and salt transport at the distal tubule in parallel with the known response of renin to these changes. Inhibition of NOS decreases renin levels at the JGA. The histochemical findings support previous concepts that MD-derived NO is involved in the control of renin synthesis.
113 citations
TL;DR: Results indicate that both PGE2 and heparin may have a role in the control of human airway smooth muscle cell growth.
Abstract: An increase in the bulk of the airway smooth muscle is a characteristic of asthma. Much of the research investigating the mechanisms of this increase in muscle has focused on mediators that are mitogenic for smooth muscle, while relatively few studies have focused on mediators inhibiting mitogenesis. In this study we have examined the effects of two mediators proposed as regulators of smooth muscle proliferation, namely heparin and prostaglandin (PG) E2, on human airway smooth muscle cells in culture stimulated with 1, 2.5, 5, and 10% fetal bovine serum (FBS) and platelet-derived growth factor AB (PDGF), 50 ng/ml. PGE2 had a biphasic effect on DNA synthesis in the presence of 1% FBS, with 10(-6) M causing inhibition and 10(-7) M causing an increase in DNA synthesis. PGE2 caused inhibition of DNA synthesis in the presence of 2.5, 5, and 10% FBS. Heparin (10 and 100 U/ml) caused an inhibition of DNA synthesis induced by 1% FBS, while 100 U/ml inhibited DNA synthesis induced by 5 and 10% FBS. PGE2 (10(-8), 10(-7), and 10(-6) M) inhibited the DNA synthesis induced by PDGF, while heparin (1, 10, and 100 U/ml) had no effect. These results indicate that both PGE2 and heparin may have a role in the control of human airway smooth muscle cell growth.
104 citations
TL;DR: It is demonstrated that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines.
Abstract: There are important phenotypic differences between endothelial cells of large vessels and the microvasculature and among microvascular endothelial cells isolated from different tissues and organs In contrast to most macrovascular endothelial cells, we demonstrate that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines To determine the identity of the inducible NOS (iNOS) isoform(s) induced by cytokines, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretreated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that was identical to the corresponding portion of the murine macrophage iNOS cDNA By use of this CMEC iNOS cDNA as a probe in Northern analyses, IL-1 beta, but not IFN-gamma, increased iNOS mRNA content in CMEC, although IFN-gamma markedly potentiated iNOS induction in these cells In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone only minimally suppressed the rise in iNOS mRNA, protein abundance, or maximal iNOS enzyme activity in whole cell lysates but suppressed nitrite production by 60% in intact CMEC Dual labeling of cytokine-pretreated CMEC in primary culture with an anti-iNOS antiserum and a fluorescein-labeled lectin specific for the microvascular endothelium of rat heart (GS-1) confirmed the presence of iNOS expression in these cells iNOS was also detected in microvascular endothelium in situ in ventricular muscle from lipopolysaccharide-, but not sham-injected, rat hearts(ABSTRACT TRUNCATED AT 250 WORDS)
100 citations
TL;DR: DHEA administration has a mixed GABAA-agonistic/antagonistic effect, exerted either directly or through DHEA-induced changes in steroid metabolism, which suggests the potential clinical usefulness of D HEA in age-related dementia.
Abstract: Dehydroepi-androsterone (DHEA) exhibits various behavioral effects in mammals, at least one of which is enhancement of memory that appears to be mediated by an interaction with the gamma-aminobutyr...
100 citations
TL;DR: The effects of the inhibition of nitric oxide (NO) production on eating behavior and thermogenesis were evaluated in the present experiments and the opposite changes in oxygen consumption, temperature, and sympathetic activity of BAT that followed L-NAME injection through the two different routes were probably due to different effect of the molecule on sympathetic output.
Abstract: The effects of the inhibition of nitric oxide (NO) production on eating behavior and thermogenesis were evaluated in the present experiments. NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO production, was injected intraperitoneally or intracerebroventricularly, and food intake, oxygen consumption rate, and interscapular brown adipose tissue (BAT) temperature were evaluated in conscious rats. The firing rate of sympathetic nerves innervating interscapular BAT was recorded in urethan-anesthetized animals. L-NAME, intraperitoneally injected, decreased food intake, oxygen consumption, temperature, and firing rate of sympathetic nerves innervating interscapular BAT. Intracerebroventricular injection of L-NAME decreased food intake and enhanced oxygen consumption, temperature, and firing rate of sympathetic nerves innervating BAT. The latter changes were similar to those found after lateral hypothalamic lesions. The opposite changes in oxygen consumption, temperature, and sympathetic activity of BAT that followed L-NAME injection through the two different routes were probably due to different effects of the molecule on sympathetic output. Impaired brain production of NO, which followed intracerebroventricular L-NAME, directly increased sympathetic activity, whereas the same activity that followed intraperitoneal L-NAME was depressed by increased blood pressure, which was elicited by the impaired peripheral production of NO.
97 citations
TL;DR: At high concentrations, EPO appears to possess a fast-acting pressor effect in vitro but not in vivo, while HTN does occur with repeated EPO administration in a time-dependent and hematocrit-independent manner, suggesting that expression of the hypertensive effect of EPO in vivo involves a gradual conditioning process.
Abstract: Hypertension (HTN) is a common complication of recombinant erythropoietin (EPO) therapy, but the mechanism of the EPO-associated HTN is uncertain. In the present study we examined the effects of EPO and the vehicle alone on rat caudal artery contractile response and basal and thrombin-stimulated platelet cytosolic Ca2+ concentration ([Ca2+]i) in vitro and on blood pressure (BP) and heart rate in vivo. At high concentrations (200 U/ml) EPO caused a small but consistent contraction in the caudal artery rings (P < 0.01) without affecting the response to either angiotensin II (ANG II) or the alpha 1-agonist methoxamine. Incubation with EPO significantly increased basal platelet [Ca2+]i (P < 0.01) and augmented the thrombin-induced rise of [Ca2+]i in Ca(2+)-free medium (P < 0.05). Long-term EPO administration led to a significant elevation of BP within 2 wk regardless of whether the hematocrit was allowed to rise or was kept constant by dietary iron deficiency. In contrast, single intravenous administration of high-dose EPO (400 and 5,000 U/kg), estimated to yield plasma concentrations comparable with those employed in vitro, failed to either alter BP or modify the BP response to ANG II during a 60-min observation period. This was associated with a significant rise in plasma guanosine 3',5'-cyclic monophosphate but no discernible change in plasma atrial natriuretic peptide, suggesting enhanced nitric oxide (NO) release. Thus, at high concentrations, EPO appears to possess a fast-acting pressor effect in vitro but not in vivo. The observed discrepancy may be due to enhanced NO release with EPO administration in vivo. However, HTN does occur with repeated EPO administration in a time-dependent and hematocrit-independent manner. This suggest that expression of the hypertensive effect of EPO in vivo involves a gradual conditioning process.
TL;DR: The results suggest that oxidative endothelial damage, and not increased hydrostatic pressure, was the primary cause of the capillary leak, and that the protection provided by inhaled NO was not solely a consequence of vasodilation.
Abstract: We found that ventilation with nitric oxide (NO, 50 ppm) significantly (P 0.05) PAP at 10 min and only modestly reduced PAP at 30 and 60 min of perfusion. Our results suggest that oxidative endothelial damage, and not increased hydrostatic pressure, was the primary cause of the capillary leak, and that the protection provided by inhaled NO was not solely a consequence of vasodilation. We conclude that inhaled NO prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs, and speculate that inhaled NO has anti-inflammatory properties in addition to its ability to cause pulmonary vasodilation.
TL;DR: The interaction between actin and the Na(+-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport and be linked to binding of actin to the enzyme.
Abstract: In this report, the functional role of actin on Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity was explored. The Na(+)- and K(+)-dependent, ouabain-sensitive ATP hydrolysis mediated by rat kidney Na(+)-K(+)-ATPase increased by 74% in the presence of previously unpolymerized actin (24 microM), whereas addition of polymerized actin was without effect. Addition of actin was associated instead with an increase in the affinity of the Na(+)-K(+)-ATPase for Na+ but not other enzymatic substates. A maximal stimulatory effect (296%) was observed either at an Na(+)-K(+)-ATPase:actin ratio of 1:50,000 or at lower ratios (1:625) by shifting from the E2 (K+ selective) to the E1 (Na+ selective) conformation of the enzyme. Immunoblotting of actin to the purified Na(+)-K(+)-ATPase suggested that this interaction may be linked to binding of actin to the enzyme, which was further supported by sequence analysis indicating putative actin-binding domains in the alpha-subunit of the enzyme. The interaction between actin and the Na(+)-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport.
TL;DR: Investigation of the role of substrate availability on 1,25(OH)2D3 production by peripheral blood monocytes in hemodialysis patients shows the need for high 25( OH)D3 levels to normalize serum 1,24R,25-dihydroxyvitamin D3, despite higher enzymatic activity.
Abstract: In chronic uremia, the requirement of supraphysiological doses of serum 25-hydroxyvitamin D3 [25(OH)D3] for the normalization of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels has been attributed to impaired substrate availability to renal 1 alpha-hydroxylase. Because serum 1,25(OH)2D3 can also be corrected by 25(OH)D3 supplementation in bilaterally nephrectomized patients, we examined the role of substrate availability on 1,25(OH)2D3 production by peripheral blood monocytes (PBM). In hemodialysis patients (HP), 25(OH)D3 uptake was 50% lower than normal, and the maximal velocity (Vmax) and apparent Michaelis constant (Km) for 25(OH)D3 of 1 alpha-hydroxylase were 2.7- and 4-fold above normal, respectively. When serum 1,25(OH)2D3 of HP was corrected by intravenous 1,25(OH)2D3, 25(OH)D3 uptake, Km, and Vmax returned to normal values. The effect of 25(OH)D3 supplementation was also examined. In normal adults, 25(OH)D3 administration had no effect on serum 1,25(OH)2D3 levels nor on the Km or the Vmax of PBM 1 alpha-hydroxylase but caused a 11-fold increase in serum 24R,25-dihydroxyvitamin D3[24R, 25(OH)2D3]. In HP, 25(OH)D3 therapy raised serum 1,25(OH)2D3 and reduced the Km and Vmax of PBM 1 alpha-hydroxylase, which correlated negatively with serum 1,25(OH)2D3. However, serum 24R,25(OH)2D3 only increased slightly above basal. These results demonstrate that, in HP, 1) impaired uptake of 25(OH)D3 and low affinity for substrate determine the need for high 25(OH)D3 levels to normalize serum 1,25(OH)2D3, despite higher enzymatic activity; 2) 1,25(OH)2D3 deficiency plays a role in enhanced 1,25(OH)2D3 synthesis and impaired access of 25(OH)D3 to PBM 1 alpha hydroxylase; and 3) abnormal 25(OH)D3 delivery also affects 24-hydroxylation.
TL;DR: The data are consistent with an involvement of MD NO synthesis in the early organization of the juxtaglomerular apparatus during nephrogenesis and suggest an interdependent relation with renin-producing cells.
Abstract: The presence of NO synthase (NOS) in cells of the macula densa (MD) suggests a role for arginine-derived NO in tubulovascular information transfer. To investigate the postnatal development of the neuronal isoform of NOS and of renin in the kidney, the cellular distribution of these enzymes was examined in perfusion-fixed kidneys of 2-, 6-, and 15-day-old rats at both the protein and mRNA level (n = 4 rats/group). NOS and renin and their mRNAs were localized by immunohistochemical and in situ hybridization methods. In addition, NOS levels were assessed by using NADPH diaphorase (NADPH-d) histochemistry. For quantification, the fraction of NOS- and renin-positive glomeruli as well as the number of NOS-positive MD cells was evaluated at all stages. Presence of NOS in single cells of the developing distal tubule was encountered already in the S-shaped body. Full expression of a NOS signal in MD cells was seen as soon as a glomerular urinary space was developed. Double labeling with NADPH-d and antibody to Tamm-Horsfall protein (THP) indicated mutual exclusiveness of NADPH-d-positive MD cells and neighboring THP-positive distal tubule cells at all levels of development. The relative intensity of renin status was 2 day > 6 day > 15 day, whereas NOS expression was maximal on postnatal day 6. Our data are consistent with an involvement of MD NO synthesis in the early organization of the juxtaglomerular apparatus during nephrogenesis and suggest an interdependent relation with renin-producing cells.
TL;DR: The pharmacological properties and signaling of angiotensin IV (ANG IV) receptors were studied in opossum kidney cell line OK7A, and data suggest that ANG IV induces Ca2+ influx inOK7A cells, at least partially, through the voltage-sensitive Ca2- channels.
Abstract: The pharmacological properties and signaling of angiotensin IV (ANG IV) receptors were studied in opossum kidney cell line OK7A. Saturation binding experiments with 125I-labeled ANG IV demonstrated the presence of high-affinity ANG IV binding sites in OK7A cell membranes with a dissociation constant (Kd) of 0.40 +/- 0.08 nM and a maximal amount of binding sites (Bmax) of 180 +/- 50 fmol/mg protein. In competition experiments, unlabeled ANG IV inhibited 125I-ANG IV binding biphasically: 20% of binding sites had high affinity [inhibition constant (Ki) = 0.44 +/- 0.04 nM] and 80% had low affinity (Ki = 130 +/- 10 nM). ANG III displaced 125I-ANG IV from binding sites with low affinity (Ki = 205 +/- 10 nM), and ANG II did not compete with 125I-ANG IV at concentrations up to 10 microM. The binding of ANG IV to OK7A cell membranes was significantly enhanced in the presence of 5 mM EDTA and completely blocked by 5 mM dithiothreitol. Guanosine 5'-O-(3-thiotriphosphate) inhibited the binding of 125I-ANG IV, indicating the G protein coupling of ANG IV receptors in OK7A cells. In signaling studies, ANG IV induced transient increase in intracellular calcium concentration ([Ca2+]i) from 49 +/- 3 to 280 +/- 45 nM. ANG IV failed to influence phosphoinositol metabolism, indicating that Ca2+ mobilization is not linked to ANG IV signaling. Ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid completely abolished ANG IV-induced increase in [Ca2+]i, consistent with Ca2+ influx. The voltage-sensitive Ca2+ channel blocking agents verapamil and nifedipine attenuated the effect of ANG IV on [Ca2+]i to 133 +/- 33 and 174 +/- 32 nM, respectively. These data suggest that ANG IV induces Ca2+ influx in OK7A cells, at least partially, through the voltage-sensitive Ca2+ channels.
TL;DR: In this article, the authors examined the effects of periodic changes in extracellular glucose concentration on matrix production and proliferation using three groups of cultured rat mesangial cells (MCs): 1) MCs in medium with continuous 5 mM glucose (CL), 2) MCm in medium alternating daily between 5 and 25 mM glucose, and 3) MC mixtures with continuous 25 mM glycolysis (CH).
Abstract: We examined the effects of periodic changes in extracellular glucose concentration on matrix production and proliferation using three groups of cultured rat mesangial cells (MCs): 1) MCs in medium with continuous 5 mM glucose (CL), 2) MCs in medium alternating daily between 5 and 25 mM glucose (PH), and 3) MCs in medium with continuous 25 mM glucose (CH). MCs cultured in PH for 10 days produced 329 and 110% more type III collagen protein than MCs cultured in CL and CH, respectively. MCs cultured in PH induced 31 and 14% more type IV collagen than MCs cultured in CL and CH, respectively. Extracellular glucose concentration had no effect on the amount of type I collagen produced. MCs cultured in PH or CH for 5 days also expressed increased levels of type I, III, and IV collagen mRNA compared with MCs cultured in CL. MCs cultured in PH for 8-10 days also produced significantly more DNA than MCs in CL or CH. These data suggest that the temporal pattern of exposure to high extracellular glucose plays a role in regulating matrix formation and cellular proliferation by MCs. Furthermore, periodic elevations of extracellular glucose had a greater stimulatory effect on collagen production than a sustained elevation. These results suggest that decreasing the variability of blood glucose concentration may decrease the adverse effect of elevated glucose levels on MC matrix production and the progression of diabetic glomerulopathy.
TL;DR: Investigation of the mechanism for the adaptive increase in Na(+)-P(i) cotransport in P(i)-deprived Hyp mice and normal littermates revealed that NaPi-2 protein was localized to the brush-border membrane of the proximal tubule and that both intensity of the signal and number of immunostained proximaltubules were increased in renal sections from normal and Hyp mice fed the low-P( i) diet.
Abstract: Although renal Na + -P i cotransporter gene expression is decreased in X-linked Hyp mice, the mutants do respond to P i restriction with an adaptive increase in Na + -P i cotransport maximal velocity in renal brush-border membrane vesicles. In the present study, we examined the mechanism for the adaptive increase in Na + -P i cotransport in P i -deprived Hyp mice and normal littermates, using a cDNA probe encoding a rat, renal-specific Na + -P i cotransporter (NaPi-2) and a rabbit polyclonal antibody raised against a synthetic NaPi-2-derived peptide. The low-P i diet elicited an increase in Na + -P i cotransport in normal (141 ± 13 to 714 ± 158) and Hyp mice (59 ± 6 to 300 ± 62 pmol-mg protein −1 .6 s −1 ; means ± SE, n = 3, P < 0.01) that was accompanied by an increase in brush-border membrane NaPi-2 protein, relative to ecto-5'-nucleotidase, in normal (1.0 ± 0.1 to 7.6 ± 1.5) and Hyp mice (0.3 ± 0.1 to 7.7 ± 1.4) (means ± SE, n = 4; P < 0.01). THE LOW-P i diet also elicited an increase in the abundance of NaPi-2 mRNA, relative to the 18S RNA, in normal (157 ± 9% of control diet, P < 0.05) and Hyp mice (194 ± 10% ofcontrol diet, P < 0.01). Immunohistochemistry revealed that NaPi-2 protein was localized to the brush-border membrane of the proximal tubule and that both intensity of the signal and number of immunostained proximal tubules were increased in renal sections from normal and Hyp mice fed the low-P i diet. In summary, 1) the adaptive increase in Na + -P i cotransport is the result of an increase in NaPi-2 mRNA and protein in both P i -deprived normal and Hyp mice; 2) abundance of NaPi-2 protein is similar in both mouse strains following P i deprivation, despite persistence of the P i transport defect in Hyp mice under these conditions; and 3) the increase in NaPi-2 protein far exceeds the increase in NaPi-2 mRNA, suggesting that translational and/or posttranslational mechanisms play a significant role in the adaptive response to P i restriction
TL;DR: It is demonstrated that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which was identified as "cPLA2" and this increase was abolished with inhibitors of protein kinase C.
Abstract: In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.
TL;DR: It is suggested that oxidants reduce tracheal epithelial barrier functions by damaging tight junctions and inhibiting cell proliferation, and these effects of oxidants on epithelial cells may be mediated by H2O2 rather than superoxide anion and by activation of protein kinase C.
Abstract: To examine the effects of oxidants on the airway epithelial barrier functions, human tracheal epithelial cells were cultured on porous filter membrane Glucose oxidase (GO; 10 U/ml), hydrogen peroxide (H2O2; 4 x 10(-3) M), and xanthine (5 x 10(-4) M) plus xanthine oxidase (20 mU/ml) (X-XO) significantly increased electrical conductance across epithelial membrane (G), short-circuit current (Isc) measured with Ussing's chamber methods, and [3H]mannitol flux through the cultured epithelium Increases in G and Isc induced by oxidants were significantly inhibited by catalase (1,000 U/ml) and the protein kinase C inhibitor staurosporine (10(-7) M), but superoxide dismutase (SOD; 100 U/ml) was without effect GO, H2O2, and X-XO inhibited the epithelial cell growth, [3H]thymidine incorporation by the cells, and epithelial repair of artificially produced focal epithelial defects (1-2 mm diam) on plastic vessels Catalase also inhibited effects induced by oxidants on cell growth and proliferation These results suggest that oxidants reduce tracheal epithelial barrier functions by damaging tight junctions and inhibiting cell proliferation, and these effects of oxidants on epithelial cells may be mediated by H2O2 rather than superoxide anion and by activation of protein kinase C
TL;DR: Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers.
Abstract: We have examined the subcellular distribution of Rho-related small GTP-binding proteins in the kidney. RhoA, CDC42, and Rac1 small GTP-binding proteins were found to be expressed at high levels in rat outer kidney cortex. Western blot analysis showed that these proteins were predominantly associated with brush-border and basolateral plasma membranes, with the exception of Rac1 which was localized predominantly in the mitochondria. RhoA and CDC42 were also found in the cytosol, and a small fraction was associated with cytoskeletal elements. A GDP-dissociation inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol. Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers. Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of these proteins with RhoGDI at an estimated size of approximately 45 kDa after gel-filtration chromatography. However, a second peak of RhoA eluted as a 20-kDa protein, indicating that not all RhoA is complexed to RhoGDI. Addition of RhoA- and CDC42-enriched fractions to purified membranes from kidney cortex resulted in their translocation to the membranes and their carboxyl methylation. Both processes were stimulated by guanosine 5'-O-(3-thiotriphosphate). Methylation inhibitors had no effect on the translocation of RhoA to membranes, suggesting that this covalent modification is not required for association to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: It is demonstrated that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.
Abstract: Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.
TL;DR: In this paper, the authors studied gene expression during the recovery from renal failure induced by folic acid and during the compensatory increase in renal function following uninephrectomy (UNX).
Abstract: The cellular effects of insulin-like growth factor I (IGF-I) are modified by a family of binding proteins (IGFBPs) that act as reservoirs in serum for the growth factor and are produced locally by tissues, including the kidney. Because regulation of these proteins may influence renal repair, either directly or by their interactions with IGF-I, we studied gene expression during the recovery from renal failure induced by folic acid and during the compensatory increase in renal function following uninephrectomy (UNX). Expression of IGF-I, the IGF-I receptor (IGF-IR), and all six IGFBPs was detected using an ribonuclease protection assay. IGFBP-5 was the most abundant binding protein mRNA present in kidney, whereas IGFBP-2 and -6 were the least abundant. During regeneration following folic acid-induced acute renal failure, IGF-I, IGFBP-3, and IGFBP-5 mRNAs declined in abundance approximately two- to threefold. On the other hand, IGF-IR, IGFBP-1, and IGFBP-2 were increased (approximately 2-, 6-, and 6-fold, respectively) in the first 24 h. IGFBP-1 mRNA remained elevated for at least 3 days. Despite the known increase in cellular RNA content following UNX, little difference in specific expression of mRNAs was observed. Because IGFBP-1 has been shown to stimulate cell migration and has previously been localized to the distal nephron, the site of greatest injury in the folic acid model, these data are compatible with the notion that this protein may function either directly to affect cellular repair or act as a reservoir for IGF-I under conditions of cellular damage.
TL;DR: To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), the reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNAs for NHE in microdissected MTAL, and it was found that NHE-3 could be the apical Na+/H+ exchanger, and N HE-1 could beThe basolateral isoform in the MTAL.
Abstract: The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.
TL;DR: It is concluded that PGI2 inhibits AVP-Lp by activation of a novel IP3 prostacyclin receptor and increases [Ca2+]i byactivation of an IP1 prostacy Clin receptor in the rabbit CCD.
Abstract: Prostaglandin E2 (PGE2) inhibits vasopressin-stimulated water conductivity (AVP-Lp) and inhibits Na+ reabsorption in the rabbit cortical collecting duct (CCD). Inhibition of Na+ reabsorption is mediated by increased intracellular calcium ion concentration ([Ca2+]i). Prostacyclin (PGI2) has also been shown to inhibit Na+ reabsorption in the CCD. The present studies were designed to examine the effect of the PGI2 agonist, Iloprost (ILP), on AVP-Lp and [Ca2+ in the isolated perfused rabbit CCD and to determine whether ILP activates different receptors than PGE2. ILP and PGE2 each maximally inhibited AVP-Lp equipotently at 10(-7) M. When CCDs were exposed to PGE2 and ILP simultaneously, or if PGE2 was added in the presence of ILP, inhibition of AVP-Lp was additive. Additivity was not observed if the PGI2 agonist, carbaprostacyclin (c-PGI2), was added with ILP, or if the PGE2 agonist, sulprostone, was added with PGE2, or if ILP was added to CCDs preexposed to PGE2. In fura 2-loaded CCD, ILP and PGE2 added separately increased [Ca2+]i. The response to c-PGI2 could be desensitized by prior exposure to ILP. ILP did not cause desensitization to PGE2, but PGE2 could desensitize the CCD to ILP. We conclude that PGI2 inhibits AVP-Lp by activation of a novel IP3 prostacyclin receptor and increases [Ca2+]i by activation of an IP1 prostacyclin receptor in the rabbit CCD. Functional evidence is presented that PGI2 cannot occupy PGE2 receptors and that PGE2 can occupy but cannot activate PGI2 receptors linked to inhibition of AVP-Lp.
TL;DR: It is proposed that since shifts in muscle type only occurred in soleus, this reflects the persistent requirement to withstand the force of gravity.
Abstract: Muscles of ky/ky homozygote mice exhibit neonatal muscle fiber necrosis and regeneration with subsequent motor nerve sprouting and development of a prominent kyphoscoliosis from approximately 100 days onward. Soleus and extensor digitorum longus (EDL) muscles from ky mice weighted < 50% of control muscles from age-matched NMRI mice. Maximal tetanic force was more reduced in soleus than in EDL. In EDL, the velocity constant of the force-velocity relation, maximal velocity, twitch time-to-peak, and isomyosin content were normal at all ages. The early mechanical changes seen in ky soleus muscles (47 day) were not accompanied by significant alterations in isomyosin or myosin heavy- and light-chain composition, since ky and NMRI expressed slow-twitch native myosin 2 (SM2, type I fibers) and intermediate-twitch native myosin (IM, type IIa fibers). Adult ky soleus (172 day) showed wholesale loss of IM and sole expression of SM2. This is sufficient to account for the markedly slowing of the force-velocity relation and the twitches observed in adult ky soleus. We propose that since shifts in muscle type only occurred in soleus, this reflects the persistent requirement to withstand the force of gravity.
TL;DR: Reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD.
Abstract: The SAD mouse is characterized by the expression of human SAD hemoglobin (Hb), a super S Hb with a higher tendency to polymerize than HbS due to the presence of two additional mutations, Antilles beta 23Ile and D Punjab beta 121Glu. Monovalent cation transport was studied in erythrocytes from SAD-1 (Hb SAD = 19%) and beta-thal/SAD-1 (Hb SAD = 26%) mice. Erythrocytes containing Hb SAD exhibited dehydration, increased maximal rate of Na(+)-K+ pump, unchanged Rb+ flux via the Gardos channel, and increased K(+)-Cl- cotransport. K(+)-Cl- cotransport was defined as Cl(-)-dependent (substitution with sulfamate or methanesulfonate) okadaic acid-sensitive K+ efflux. Volume regulatory decrease via K(+)-Cl- cotransport was also increased in swollen SAD erythrocytes compared with controls. K(+)-Cl- cotransport was stimulated by staurosporine in all mouse strains, but the extent of stimulation was reduced in beta-thal/SAD-1 mice. Treatment with dithiothreitol reduced K(+)-Cl- cotransport activity in SAD-1 and beta-thal/SAD-1 mice to levels similar to that of control strains, indicating that reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD.
TL;DR: The data indicate that flounder possess an active mechanism for the renal excretion of Dau that is stimulated by mild heat shock and has characteristics consistent with transport by an apical P-glycoprotein.
Abstract: Primary monolayer cultures of winter flounder renal proximal tubule epithelium mounted in Ussing chambers were used to characterize transepithelial transport of daunomycin (Dau). Control tissues performed active net secretion of Dau (0.064 +/- 0.027 nmol.cm-2.h-1). Mild heat shock (5 degrees C elevation for 6-8 h followed by return to normal temperature) almost doubled Dau secretion (0.114 +/- 0.026 nmol.cm-2.h-1). This response was inhibited approximately 40% by addition of the protein synthesis inhibitor, cycloheximide. Dau secretion was inhibited by verapamil, vinblastine, cyclosporin A, and to a lesser degree by the organic cation, tetraethylammonium. In addition, tetraethylammonium secretion was inhibited by vinblastine. Dau secretion was not inhibited by the organic anion, p-aminohippurate, and p-aminohippurate secretion was not inhibited by vinblastine. The transepithelial reabsorptive flux of Dau and the electrical characteristics of the tissues, including rheogenic glucose transport, were unaffected by any of the above treatments. Reaction of tissues with a monoclonal antibody to P-glycoprotein (C219) revealed the presence of this transporter on only apical microvilli. The data indicate that flounder possess an active mechanism for the renal excretion of Dau that is stimulated by mild heat shock. This mechanism is distinct from organic anion, but not organic cation, transport and has characteristics consistent with transport by an apical P-glycoprotein.
TL;DR: Apolipoprotein AI (apo AI) synthesis, measured as the turnover of 125I-labeled apo AI-labeling high-density lipoprotein (HDL), was increased significantly in rats with Heymann nephritis vs. control Sprague-Dawley (SD) rats, but fractional apoAI catabolic rate was also significantly less in HN vs. SD.
Abstract: Apolipoprotein AI (apo AI) synthesis, measured as the turnover of 125I-labeled apo AI-labeled high-density lipoprotein (HDL), was increased significantly in rats with Heymann nephritis (HN) vs. control Sprague-Dawley (SD) rats. However, fractional apo AI catabolic rate was also significantly less in HN vs. SD. We used 125I-apo AI tyramine cellobiose HDL, a marker retained at the catabolic site, to establish where apo AI catabolism decreased in six HN rats, seven rats with adriamycin (Adria)-induced nephrosis, and six control SD. Total renal apo AI catabolism, plus urinary losses, were the same in all three groups, despite significant urinary apo AI in HN and Adria rats. Apo AI catabolism was reduced in skin in both nephrotic groups, accounting for approximately 44% of reduced in apo AI catabolism. Thus a significant fraction of apo AI is catabolized in skin of normal male rats. Reduced apo AI catabolism in skin contributes to increased plasma levels in nephrotic rats.