Isolation and characterization of methane utilizing bacteria from wetland paddy ecosystem.

Methylotrophic bacteria which are known to utilize C1 compounds including methane. Research during past few decades increased the interest in finding out novel genera of methane degrading bacteria to efficiently utilize methane to decrease global warming effect. Moreover, evaluation of certain known plant growth promoting strains for their methane degrading potential may open up a new direction for multiple utility of such cultures. In this study, efficient methylotrophic cultures were isolated from wetland paddy fields of Gujarat. From the overall morphological, biochemical and molecular characterization studies, the isolates were identified and designated as Bacillus aerius AAU M 8; Rhizobium sp. AAU M 10; B. subtilis AAU M 14; Paenibacillus illinoisensis AAU M 17 and B. megaterium AAU M 29. Gene specific PCR analysis of the isolates, P. illinoisensis, B. aerius, Rhizobium sp. and B. subtilis showed presence of pmoA gene encoding α subunit particulate methane monooxygenase cluster. B. megaterium, P. illinoisensis, Rhizobium sp. and Methylobacterium extrorquens showed presence of mmoX gene encoding α subunit of the hydroxylase component of the soluble methane monooxygenase cluster. P. illinoisensis and Rhizobium sp. showed presence mxaF gene encoding α subunit region of methanol dehydrogenase gene cluster showing that both isolates are efficient utilizers of methane. To the best of our knowledge, this is the first time report showing presence of methane degradation enzymes and genes within the known PGPB group of organisms from wet land paddy agro-ecosystem, which is considered as one of the leading methane producer.

CD1d-associated expression of NF-kB and cardiac dysfunction in diabetic and obese mice.

In patients with obesity and diabetes mellitus, abnormal production of inflammatory factors may result in cardiovascular dysfunction. In the current study, we tested the impact of CD1d-mediated innate immune responses on the expression and activation of NFkB in the hearts of adipose diabetic (db/db) mice. Splenocytes from adult db/db and CD1d-knockout mice of both genders and their wild-type, C57BL/6 and Balb/C counterparts were examined for tumor necrosis factor (TNF)-alpha and TNF-alpha receptor type 1. The percentage of natural killer T (NKT) cells in CD3+ T cells was compared with that in nondiabetic control mice. Despite the absence of inflammatory infiltrates, the hearts of db/db mice showed alterations in TNF-alpha receptor-1 and NFkB activity, including increased expression of both the NFkB p52 and p65 subunits. In the hearts of CD1d-knockout mice, p52 expression was reduced, while p65 expression remained largely unchanged. On echocardiography, the ratio of E to A transmitral flow velocities (an indicator of diastolic function) was significantly decreased in db/db mice after they swam for 30 minutes. These results provide evidence for CD1d-mediated NFkB activation and diastolic dysfunction in the hearts of db/db mice. Therefore, CD1d-associated abnormalities of innate immune responses and TNF-alpha production in splenic tissue may contribute to NFkB activation and cardiac dysfunction in type 2 diabetes.

Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries.

Tissue factor (TF), the initiator of blood coagulation and thrombosis, is up-regulated after vascular injury and in atherosclerotic states. Systemic administration of recombinant TF pathway inhibitor (TFPI) has been reported to decrease intimal hyperplasia after vascular injury and also to suppress systemic mechanisms of blood coagulation and thrombosis. Here we report that, in heritable hyperlipidemic Watanabe rabbits, adenoviral gene transfer of TFPI to balloon-injured atherosclerotic arteries reduced the extent of intimal hyperplasia by 43% (P < 0.05) compared with a control vector used at identical titer (1 x 10(10) plaque-forming units/ml). Platelet aggregation and coagulation studies performed 7 days after local gene transfer of TFPI failed to show any impairment in systemic hemostasis. At time of sacrifice, 4 weeks after vascular injury, the 10 Ad-TFPI treated carotid arteries were free of thrombi, whereas two control-treated arteries were occluded (P, not significant). These findings suggest that TFPI overexpressed in atherosclerotic arteries can regulate hyperplastic response to injury in the absence of changes in the hemostatic system, establishing a role for local TF regulation as target for gene transfer-based antirestenosis therapies.

Growth suppression of human coronary vascular smooth muscle cells by gene transfer of the transcription factor E2F-1.

BACKGROUND: The transcription factor E2F-1 promotes S-phase entry and death in transformed cells and primary cardiomyocytes. We tested the hypothesis that overexpression of E2F-1 forces growth-arrested human coronary vascular smooth muscle cells (VSMCs) to enter the S phase, undergo apoptosis, and thereby regulate VSMC growth. METHODS AND RESULTS: Early-passage (8 times. CONCLUSIONS: Overexpression of the transcription factor E2F-1 regulates growth of human coronary VSMCs by forcing the cells to enter the S phase and then to die. Cell death appears to involve caspase 3-like activity, which, in the VSMCs, is markedly increased by overexpression of E2F-1.

Thromboresistance of balloon-injured porcine carotid arteries after local gene transfer of human tissue factor pathway inhibitor.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of factor Xa and the coagulant initiator complex tissue factor/factor VIIa. METHODS AND RESULTS: To study the effects of TFPI gene transfer on thrombus formation, balloon-injured porcine carotid arteries were treated locally with an adenovirus encoding human TFPI (Ad-TFPI) or control virus. Gene transfer of TFPI was confirmed by detection of human TFPI in the conditioned medium of porcine carotid arteries kept in culture after in vivo transduction. When carotid flow was measured with Doppler probe 5 days after surgery, cyclic flow variations (CFVs) developed in 7 of 8 control pigs after constriction of the injured carotid artery by 40%, and all control-treated arteries occluded after 70% constriction. In contrast, CFVs were observed in only 1 of 8 Ad-TFPI-treated pigs after 40% constriction, and only 3 of 8 occluded after constriction by 70% (P=0.0027 and P=0.007, respectively). None of the 5 TFPI-transduced arteries open after 70% constriction developed CFVs during an incremental epinephrine infusion. CONCLUSIONS: Compared with baseline, systemic hemostatic variables and platelet aggregation were unimpaired, suggesting that TFPI gene transfer can prevent arterial thrombosis in the presence of severe shear stress and without detectable hemostatic impairment.

[New antithrombotic therapy approaches in coronary heart disease--prospects for gene therapy]. / Neue antithrombotische Therapieansätze bei koronarer Herzerkrankung--Perspektiven einer Gentherapie.

Despite considerable progress, pharmacological therapies have not provided a complete solution for common cardiovascular problems, including recurrent thrombosis, restenosis, and vein graft deterioration. Optimal drug dosage, reproducing plasma concentrations achieved in animal studies establishing proof-of-principle, would often be too toxic to administer. Local gene therapy aims at overexpressing proteins that regulate the cell cycle of vascular smooth muscle cells, inhibit vascular smooth muscle cell migration, endow the endothelium with enhanced vasoprotective properties. Alternatively, some approaches tend to suppress gene expression of proteins believed to promote vascular smooth muscle cell proliferation and migration. In sharp contrast to drug treatments, local gene therapy limits expression of the beneficial agent to the injured vascular site, where it can extend the presence of this agent to weeks and, with some gene vectors, to many months. This review summarizes and discusses antithrombotic gene therapy approaches for the prevention of restenosis and late thrombosis after catheter-based revascularizations.

The emerging clinical potential of cardiovascular gene therapy.

Despite considerable progress, pharmacological therapies have not provided a complete solution for common cardiovascular problems, including recurrent thrombosis, restenosis, and vein graft deterioration. Optimal drug dosage, reproducing plasma concentrations achieved in animal studies establishing proof-of-principle, would often be too toxic to administer, especially when given over prolonged periods of time. Local gene therapy aims at overexpressing proteins that: (1) regulate the cell cycle of VSMC; (2) inhibit VSMC migration; (3) endow the endothelium with its vasoprotective properties; and (4) stimulate growth of endothelium and angiogenesis. Alternatively, some approaches tend to suppress gene expression of proteins believed to promote VSMC proliferation and migration. In sharp contrast to drug treatments, local gene therapy limits expression of the beneficial agent to the injured vascular site, and there, it can extend the presence of this agent to weeks and, with some gene vectors, to many months. The clinical potential of this approach has led to the initiation of trials that currently evaluate gene therapy approaches to the attenuation of peripheral and myocardial ischaemia and the prevention of vein graft disease.

Short-chain fatty acid (SCFA) volume regulation in proximal and distal rabbit colon is different.

SCFAs increase the volume of many different cell types rarely exposed to significant concentrations of these weak electrolytes. SCFAs swell isolated cells from colonic carcinoma cell lines, but the-mechanism(s) of volume regulation in normal colonocytes, which are generally exposed to > 100 mM SCFAs, has not been well characterized. AIMS. To determine the effect of SCFAs on volume regulation in proximal and distal rabbit colonocytes. METHODS. Isolated colonocytes were plated on coverslips and placed in a perfusion apparatus that permitted fluid changes. Cells were continuously monitored by video-microscopy; volume was estimated by measured changes in the radius of individual cells. RESULTS. Distal colonocytes (DC) consistently had a slightly greater basal volume than proximal colonocytes (PC): [14.2 pl/fl:9.8 pl/fl] In HEPES-buffered solutions, an isotonic change to a 90 mM NaCl/50 mM Na propionate solution elicited a significant increase in cell volume within 10 min, but no noticeable regulatory volume decrease over 30 min: V/Vo in DC: 1.29 +/- .09; in PC: 1.25 +/- .05. In HCO3-buffered solutions, 50 mM PROP caused significantly greater cell swelling; in DC: 1.74 +/- .21; in PC: 1.52 +/- .08. In DC both amiloride and EIPA blocked the SCFA-induced increase in cell volume. A hypotonic challenge confirmed that these cells were capable of swelling. In contrast, amiloride did not significantly inhibit SCFA-induced swelling in PC: control, 1.25 +/- .05; amiloride, 1.36 +/- .10. Cell volume increased in PC perfused with an isosmotic 50 mM propionate, Na-free solution: 1.22 +/- .04. CONCLUSIONS. (i) SCFAs induce significant cell swelling, but no regulatory volume decrease, in isolated colonocytes; (ii) HCO3 augments SCFA-induced cell swelling; (iii) volume increase in DC is dependent on Na-H exchange, but in PC appears to be Na-independent. SIGNIFICANCE. There are fundamental differences in how proximal and distal colon respond to isosmotic volume challenge of SCFAs.

Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle.

Resistance to insulin-induced glucose disposal is associated with hypertension, in accord with recent reports that insulin-induced vasodilation is impaired in men with resistance to insulin-induced glucose disposal. Nevertheless, the mechanism of insulin-induced vasodilation is not known. We wished to determine whether a physiological concentration of insulin inhibits agonist-induced contraction at the level of the individual vascular smooth muscle cell, and if so, how. Dispersed vascular smooth muscle cells from dog femoral artery were grown on collagen gels for 4 to 8 days. Contraction and intracellular Ca2+ concentration of individual cells were measured by photomicroscopy and fura 2 epifluorescence microscopy, respectively. Serotonin and angiotensin II contracted cells in a dose-dependent manner. Preincubation of cells for 20 minutes (short-term) or 7 days (long-term) with insulin (40 microU/mL) inhibited serotonin- and angiotensin II-induced contractions by approximately 50%. Insulin (10 microU/mL) acutely inhibited serotonin-induced contraction by 34%. The maximal effect of high extracellular K(+)-induced contraction was not affected by short-term insulin exposure, but the ED50 for extracellular K(+)-induced contraction was increased from 7.6 +/- 2.5 to 16.0 +/- 3.9 mmol/L (P < .05). Short-term insulin exposure also attenuated the peak rise of the serotonin-induced intracellular Ca2+ transient and increased the rate constant for intracellular Ca2+ decline. Verapamil and ouabain completely blocked the attenuation of agonist-induced contraction by short-term insulin exposure, indicating the importance of voltage-operated Ca2+ channels and the Na(+)-K+ pump for this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of serotonin on intracellular pH and contraction in vascular smooth muscle.

Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.

Effects of pHi on Na(+)-H+, Na(+)-dependent, and Na(+)-independent C1(-)-HCO3-exchangers in vascular smooth muscle.

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. We reported that pHi in primary cultured vascular smooth muscle cells from canine femoral artery is 7.26, a value maintained via HCO3- influx by the Na(+)-dependent C1(-)-HCO3-exchanger but not via H+ efflux by the Na(+)-H+ exchanger [A. M. Kahn, E. J. Cragoe, Jr., J. C. Allen, R. D. Halligan, and H. Shelat. Am. J. Physiol 259 (Cell Physiol. 28): C134-C143, 1990]. To explain these findings, in the present study, we determined the pHi activity profile of these two transport systems. Although both were active at acidic pHi, Na(+)-H+ exchange activity was very low at and above pHi 7.0, while Na(+)-dependent C1(-)-HCO3-exchange activity maintained near-maximal activity up to pHi 7.26 but fell to undetectable levels by pHi 7.4. A Na(+)-independent C1(-)-HCO3-exchanger was present, which mediated HCO3-efflux after an acute alkaline load. The activity of this system was negligible at pHi 7.2 and was stimulated at alkaline pHi. In conclusion, the pHi of these vascular smooth muscle cells at rest is maintained via HCO3-influx by the Na(+)-dependent C1(-)-HCO3-exchanger. After acute acidic or alkaline loads, correction of pHi is mediated by activation of the normally quiescent Na(+)-H+ and Na(+)-independent C1(-)-HCO3-exchangers, respectively. All three acid-base transport systems have pHi set points that protect the cell from overcorrecting pHi after a disturbance in either direction.

Na(+)-independent Cl(-)-HCO3- exchange in sarcolemmal vesicles from vascular smooth muscle.

Intracellular pH (pHin) affects vascular smooth muscle function, but the mechanisms that control pHin in this tissue are not well understood. These studies were performed to determine whether sarcolemmal vesicles from bovine superior mesenteric artery (SMA) contain a Na(+)-independent Cl(-)-HCO3- exchanger and, if so, to determine its sensitivity to membrane voltage and inhibitors. 36Cl- was taken up by vesicles into an osmotically active intravesicular space. In Na(+)-free media, an outwardly or inwardly directed HCO3- gradient stimulated 36Cl- transport in the opposite direction. An outwardly directed unlabeled Cl- gradient stimulated 36Cl- uptake by a mechanism that was inhibited by external HCO3-. HCO3- or Cl- gradient-stimulated 36Cl- uptake was not due to voltage coupling between ions. In the nominal absence of HCO3-, a threefold outwardly directed OH- gradient did not affect 36Cl- uptake. Total 36Cl- uptake was stimulated by an inside-positive voltage, but the HCO3- gradient-stimulated component of 36Cl- uptake was insensitive to a change in membrane voltage. Finally, HCO3- gradient-stimulated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and furosemide, with 50% inhibitory concentration values equalling approximately 1.0 and 0.5 mM, respectively. These data indicate that sarcolemmal vesicles from bovine SMA contain a Na(+)-independent Cl(-)-HCO3- exchanger. This transport system is probably electroneutral and is inhibitable by DIDS and furosemide. A conductive pathway for Cl- is present in the vesicles, but Cl(-)-OH- exchange activity was not observed.

Na(+)-H+ and Na(+)-dependent Cl(-)-HCO3- exchange control pHi in vascular smooth muscle.

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. These studies were performed to determine the identity and relative importance of the sarcolemmal transport systems that mediate net acid efflux in primary cultured vascular smooth muscle cells from canine femoral artery. In HEPES- or HCO3(-)-buffered physiological salt solution (HEPES-PSS, HCO3(-)-PSS), recovery from an acute acid load was totally dependent on external Na+. 5-[N-ethyl-N-isopropyl]amiloride (EIPA, 50 microM) inhibited pHi recovery 100 and 68% in HEPES-PSS and HCO3(-)-PSS, respectively. EIPA-insensitive pHi recovery in HCO3(-)-PSS was inhibited 48% by 4,4'-diisothyocyanostilbene-2,2'-disulfonic acid (DIDS). An outwardly directed H+ gradient stimulated amiloride-sensitive 22Na+ uptake, and an inwardly directed HCO3- gradient stimulated amiloride-insensitive 22Na+ uptake. The latter was inhibited by DIDS or prior depletion of cell Cl-. In HEPES-PSS, resting pHi was 7.17 +/- 0.03, was not affected by DIDS, but was lowered by EIPA or by removing extracellular Na+. In HCO3(-)-PSS, resting pHi was 7.25 +/- 0.02 (P less than 0.05) and was not affected by EIPA. Removing extracellular Na+ in the presence of EIPA decreased pHi in HCO3(-)-PSS but not in HEPES-PSS. DIDS lowered resting pHi in HCO3(-)-PSS, after which EIPA further lowered pHi. We conclude that acid efflux from these cells is mediated by a Na(+)-H+ exchanger and a Na(+)-dependent Cl(-)-HCO3- exchanger. In HEPES-PSS, acid efflux via the Na(+)-H+ exchanger maintains resting pHi. In HCO3(-)-PSS, additional acid efflux via the Na(+)-dependent Cl(-)-HCO3- exchanger results in a higher pHi. Although the Na(+)-H+ exchanger is primarily responsible for acid efflux after an acute acid load, the Na(+)-dependent Cl(-)-HCO3- exchanger is responsible for acid efflux under physiological conditions.

Sodium-lithium exchange and sodium-proton exchange are mediated by the same transport system in sarcolemmal vesicles from bovine superior mesenteric artery.

Several laboratories have reported that Na+-Li+ countertransport activities are increased in red blood cells from patients with essential hypertension. It has been proposed that the activity of this red blood cell transport system might reflect the activity of a similar system in vascular smooth muscle. We previously demonstrated Na+-Li+ exchange in sarcolemmal vesicles from canine artery and proposed that this transport function might be mediated by the Na+-H+ exchanger. In the present studies, however, we were unable to demonstrate Na+-Li+ countertransport in canine red blood cells. Since bovine red blood cells have a vigorous Na+-Li+ exchanger and we previously demonstrated Na+-H+ exchange in sarcolemmal vesicles from bovine artery, we wished to determine whether bovine sarcolemmal vesicles mediate Na+-Li+ exchange and whether this transport function is mediated via the Na+-H+ exchanger. We found that an outwardly directed proton or Li+ gradient stimulated 22Na+ uptake in sarcolemmal vesicles from bovine superior mesenteric artery. Li+ gradient-stimulated Na+ uptake was not due to electrical coupling between the two ions, was not affected by a change in membrane potential, and could not be explained by the parallel operation of Li+-H+ and Na+-H+ exchange. External Li+ inhibited proton gradient-stimulated Na+ uptake, and external protons inhibited Li+ gradient-stimulated Na+ uptake. Na+ efflux from vesicles was stimulated by inwardly directed gradients for Li+ or protons, and these effects were not additive. Proton efflux from vesicles was stimulated by inwardly directed gradients for Na+ or Li+, and these effects were not additive. Finally, Na+-H+ exchange and Na+-Li+ exchange in sarcolemmal vesicles were inhibited by 5-(N-ethyl-N-isopropyl)amiloride in an identical dose-dependent manner. In conclusion, Na+-Li+ countertransport could not be demonstrated in canine red blood cells, but as is the case with bovine red blood cells, sarcolemmal vesicles from bovine artery mediate Na+-Li+ countertransport. This transport function and sarcolemmal Na+-H+ exchange are mediated via a single 5-(N-ethyl-N-isopropyl)amiloride-sensitive cation exchanger with affinity for Na+, Li+, and protons. The cow, as opposed to the dog, may be a good animal model to test whether the activity of red blood cell Na+-Li+ countertransport is predictive of the activity of Na+-Li+ (and Na+-H+) exchange in vascular smooth muscle.

Sodium-lithium exchange in sarcolemmal vesicles from canine superior mesenteric artery.

Exchange of intracellular sodium for extracellular lithium readily occurs in vascular smooth muscle, but the mechanism of this exchange is not known. These studies examined whether a sodium-lithium countertransport system was present in the cell membrane of vascular smooth muscle. A sarcolemmal-enriched vesicle preparation was obtained from canine superior mesenteric artery via a magnesium aggregation and differential centrifugation technique. An outwardly directed gradient for lithium stimulated 22Na uptake by the vesicles, and an inwardly directed gradient for lithium stimulated 22Na efflux. These effects were not due to an alteration in membrane potential, and sodium uptake was not stimulated by lithium in the absence of a gradient for lithium. The lithium gradient-stimulated component of sodium uptake was not affected by a change in membrane potential and was insensitive to ouabain. Both sodium-lithium exchange and sodium-proton exchange in sarcolemmal-enriched vesicles were inhibited by two compounds that inhibit the sodium-lithium countertransport system in red cells, phloretin and quinidine. Ethylisopropylamiloride also inhibited both sodium-lithium exchange and sodium-proton exchange in the vesicles. In support of the possibility that sarcolemmal sodium-lithium exchange and sodium-proton exchange are mediated by a single cation exchange mechanism with affinity for sodium, lithium, and protons, we found that an inwardly directed sodium or lithium gradient stimulated proton efflux, and that the stimulation of sodium efflux by external lithium or protons was not additive. It is concluded from these studies that sarcolemmal vesicles from canine superior mesenteric artery contain an electroneutral, phloretin, quinidine, and ethylisopropylamiloride inhibitable sodium-lithium exchange transport system.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+-Ca2+ exchange in sarcolemmal vesicles from bovine superior mesenteric artery.

These studies were designed to determine whether a Na+-Ca2+ exchanger is present in sarcolemmal vesicles from bovine superior mesenteric artery and, if so, to determine whether this transport system is qualitatively similar to that found in other excitable tissues. Vesicles, preferentially enriched in sarcolemma, were prepared by a Mg2+ aggregation and differential centrifugation technique. An inwardly directed Ca2+ gradient stimulated 22Na+ efflux and an outwardly directed Ca2+ gradient stimulated 22Na+ uptake. Similarly, an inwardly directed Na+ gradient stimulated 45Ca2+ efflux, and an outwardly directed Na+ gradient stimulated 45Ca2+ uptake. Ca2+ gradient-stimulated Na+ transport and Na+ gradient-stimulated Ca2+ transport were not due to voltage coupling between the two ions. Hence, a Na+-Ca2+ exchanger is present in these vesicles. The Na+ gradient-dependent component of Ca2+ uptake (Na+-Ca2+ exchange) was stimulated by rendering the vesicles electropositive inside, and Na+-Ca2+ exchange activity was inhibited by amiloride and quinidine in a dose-dependent fashion. These data demonstrate similarities between this mesenteric arterial smooth muscle Na+-Ca2+ exchanger and that found in other excitable tissues. In the absence of added Ca2+, amiloride-sensitive 22Na+ uptake in the vesicles was stimulated by an outwardly directed proton gradient, and an inwardly directed Na+ gradient stimulated proton efflux. Thus these vesicles also contain a Na+-H+ exchanger, which has been found in the sarcolemma of other vascular smooth muscle cells. When Na+ uptake was stimulated via Na+-H+ exchange, the subsequent uptake of Ca2+ via Na+-Ca2+ exchange was tripled. In conclusion, these studies unequivocally demonstrate that sarcolemmal-enriched vesicles from bovine superior mesenteric artery contain a Na+-Ca2+ exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+-H+ exchange is present in sarcolemmal vesicles from dog superior mesenteric artery.

The Na+ concentration inside vascular smooth muscle cells is an important regulator of vascular smooth muscle function, but the mechanisms that mediate Na+ influx are not known. We studied Na+ transport in a newly described vesicle preparation preferentially enriched in sarcolemma and obtained by Mg2+ aggregation and differential centrifugation of homogenized dog superior mesenteric artery. In the presence of an outwardly directed proton gradient (pHout = 7.5, pHin = 5.0), 1 mM 22Na+ uptake was stimulated over twofold relative to the absence of a pH gradient (pHin = 7.5 or 5.0). pH gradient-stimulated Na+ uptake was inhibited by 1 mM amiloride. 22Na efflux was stimulated by an inwardly directed proton gradient (pHin = 7.5, pHout = 5.7 vs. 7.5). The rate of proton efflux from acid-loaded vesicles was measured by acridine orange fluorescence and was stimulated by 100 mM Naout but not by Nain = Naout = 100 mM. H+ gradient-stimulated Na+ transport and Na+ gradient-stimulated H+ transport were not due to electrical coupling between the two cations. The pH gradient-stimulated component of Na+ transport in the final vesicles, an intermediate fraction, and microsomes were proportional to the respective enzyme marker activities for sarcolemma but not for sarcoplasmic reticulum or mitochondrial membranes. We conclude that Mg2+ aggregation and differential centrifugation of homogenized vascular smooth muscle yield a vesicle preparation preferentially enriched in sarcolemma. Furthermore, the sarcolemma of vascular smooth muscle contains an amiloride-sensitive Na+-H+ proton countertransport system.

Urate and p-aminohippurate transport in rat renal basolateral vesicles.

We examined the transport of urate in basolateral membrane vesicles from the rat kidney and determined the relationship between the transport of urate and p-aminohippurate (PAH). Urate was not converted to allantoin, and the uptake of urate represented transport into an osmotically active intravesicular space. The 10-s uptake of 53 microM [14C]urate in basolateral vesicles was inhibited 39 +/- 6, 49 +/- 10, and 35 +/- 3% by (in mM) external 2.4 probenecid, 2.4 DIDS, and 1.4 unlabeled urate, respectively. The 10-s uptake of 353 microM [14C]urate was trans-stimulated 82 +/- 8% by preloading basolateral vesicles with 1.5 mM unlabeled urate. The uptake of urate was stimulated by an outwardly directed gradient for Cl- (Cl-in = 25 mM, Cl-out = 5 mM). This effect was not consequent to a more electropositive intravesicular space, as monitored by the voltage-sensitive sodium-L-malate cotransport system. The Cl- gradient-stimulated component of urate uptake in basolateral vesicles was not cis-inhibited by 4.8 mM PAH, whereas Cl gradient-stimulated urate uptake in brush border vesicles was cis-inhibited 43 +/- 5% by PAH. In the absence of Cl-, 4.8 mM PAH did not cis-inhibit, and 5.4 mM PAH or 6.4 mM lactate did not trans-stimulate the uptake of urate in basolateral vesicles, contrasting with results obtained with brush border vesicles. The uptake of urate in basolateral vesicles was not stimulated by external Na+ relative to K+, Li+, or Cs+. In contrast, PAH uptake in basolateral vesicles was stimulated 87 +/- 9% by external Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

Age affects dopamine receptor changes during chronic administration of fluphenazine.

The effects of age on the alteration of brain dopaminergic receptors (spiperone binding) during chronic administration of fluphenazine was studied in young (7-month) and old (25-month) rats. Compared to age-matched saline controls, the old fluphenazine rats showed an earlier on-set and quantitatively greater decrease in specific binding of spiperone and increase in the KD of spiperone binding than young rats. Young rats showed an increase in the Bmax of spiperone binding at 6-weeks which was not shown by old rats.

Influence of age on the effects of chronic fluphenazine on receptor binding in rat brain.

The effects of age on alterations in brain dopaminergic (spiperone), beta-adrenergic (DHA), alpha-adrenergic (WB-4101), and cholinergic (QNB) binding induced by chronic administration of fluphenazine was studied in the rat. Compared to age-matched saline controls, older-age (25 month) fluphenazine rats showed: (a) a similar percent increase in specific spiperone binding (at 0.1 nM) but a slightly smaller increase in the Bmax of spiperone binding in the striatum than younger fluphenazine-treated rats; (b) a substantially greater increase in the Bmax of DHA binding and a slightly greater increase in Bmax of WB-4101 binding in the cerebral cortex than younger fluphenazine-treated rats. There was no significant interaction of age with the effect of chronic fluphenazine on QNB binding in rat striatum.