Biomedical subjects

J Kolls

Publications and source records attributed to J Kolls.

26 records · Page 2Linked to original sources

PubMed1994

Rapid induction of messenger RNA for nitric oxide synthase II in rat neutrophils in vivo by endotoxin and its suppression by prednisolone.

Nitric oxide is believed to participate in nonspecific cellular immunity. Gram negative bacterial endotoxins increase the production of reactive nitrogen intermediates (RNI) in phagocytic cells by inducing the enzyme nitric oxide synthase II (NOS II). Anti-inflammatory glucocorticoids attenuate endotoxin-induced increases in RNI. This study evaluated the effect of in vivo administration of prednisolone on Escherichia coli lipopolysaccharide endotoxin (LPS)-induced increases in plasma RNI and neutrophil mRNA for NOS II and production of RNI in the rat. We show that LPS rapidly induces mRNA for NOS II and production of RNI (NO2- and NO3- anion) in rat neutrophils within 2 hr after in vivo administration of a sublethal dose of 0.5 mg/kg, i.v. A pharmacologic dose of prednisolone (50 micrograms/kg, im) given 15 min before LPS-attenuated production of NO2- and NO3- by neutrophils and suppressed LPS-stimulated mRNA for NOS II. 3-Amino, 1,2,4-triazine inhibited NO2- and NO3- production without affecting gene expression for NOS II. These data demonstrate that LPS rapidly induces functional gene expression for NOS II and prednisolone prevents induction of NOS II activity by inhibiting transcription of its mRNA.

Amino Acid Oxidoreductases↗

PubMed1993

Alcohol administration attenuates LPS-induced expression of inducible nitric oxide synthase in Kupffer and hepatic endothelial cells.

This study investigates the effects of in vivo ethanol (primed infusion, causing 170-190 mg% plasma alcohol for 12 hours) and/or LPS (12 hours after injection of E. coli LPS 1 mg/kg bw.) on the mRNA expression of inducible nitric oxide synthase (NOS II) in hepatic cells measured by competitive PCR technique, and on hepatic release of reactive nitrogen intermediates (RNI, NO2- + NO3-). Perfused livers from alcohol- or saline-infused animals did not release measurable amounts of RNI. Under these conditions small amounts of NOS II mRNA were expressed in Kupffer and endothelial cells, while it was not detectable in parenchymal cells. LPS treatment along with markedly elevating hepatic RNI release increased NOS II mRNA levels by 35- and 200-fold, in endothelial and Kupffer cells, respectively. LPS injection and alcohol infusion to the same animal decreased hepatic RNI release by about 70% and almost completely inhibited the LPS-induced, elevated NOS II mRNA in Kupffer or endothelial cells. No similar changes were observed in the parenchymal cells. These data suggest that the primary target of in vivo LPS in upregulating hepatic NO release are the nonparenchymal cells. Furthermore, alcohol inhibits the LPS-induced response which may influence immune-related hepatic function.

Amino Acid Oxidoreductases↗

PubMed1993

cDNA equalization for reverse transcription-polymerase chain reaction quantitation.

Reverse transcription coupled with the polymerase chain reaction has been increasingly utilized to study gene expression. However, most previously published quantitative techniques are limited by accurate initial RNA quantitation and do not account well for the relative efficiency of reverse transcription. We have developed a technique of labeling and quantitating the random-primed cDNA product of a reverse transcription reaction. Using the polymerase chain reaction in conjunction with template dilutions or with an internal competitive template, we show that by normalizing the cDNA input into the polymerase chain reaction, we get accurate quantitation of gene expression. We call this procedure cDNA equalization of reverse transcriptase-polymerase chain reaction. This method is ideal for small clinical samples where accurate quantitation of input RNA is difficult.

Animals↗

PubMed1993

Tumor necrosis factor-alpha inhibits endothelium-dependent relaxation.

Tumor necrosis factor-alpha (TNF-alpha) stimulates nitric oxide (NO) in vascular endothelium by induction of the enzyme NO synthase II (NOS II). We examined the effects of TNF-alpha on 1) endothelium-dependent (EDR) and endothelium-independent (EIR) relaxation and 2) contraction of bovine intralobar pulmonary arteries (BPA) and veins (BPV) in vitro. Acetylcholine (ACh), bradykinin (BK), histamine, and A23187 produced EDR of BPA contracted with a 50% effective concentration of U-46619 (15 nM), because relaxation was abolished by endothelium-rubbing and attenuated by L-NG-mono-methylarginine (L-NMMA; 300 microM). TNF-alpha (0.00417, 0.0417, 0.417, and 1.25 micrograms/ml) incubated with BPA for 60 min inhibited EDR of the BPA to ACh, BK, and histamine. The effects of TNF required 30 min for onset. Recovery of EDR occurred 3-4 h after washout of TNF-alpha. Pentoxifylline (1 microM) did not affect ACh-induced EDR but selectively reversed TNF-alpha-mediated inhibition of ACh-induced EDR. TNF-alpha-mediated inhibition of EDR was not reversible by L-NMMA, an inhibitor of NOS I and NOS II, the cyclooxygenase inhibitor ibuprofen, or CV-3908 (1 microM), a platelet-activating factor antagonist. The inhibitory effect of TNF-alpha on EDR was not mediated by nonspecific sensitization of the endothelium to human protein because recombinant human granulocyte colony-stimulating factor (10, 50, and 500 x 10(3) U/ml) did not affect EDR of BPA. The effect of TNF-alpha was specific for release of NO from the endothelium of BPA because TNF-alpha did not affect 1) EDR of BPV to ACh, BK, or ATP; 2) EIR of BPA or BPV to nitroprusside; and 3) contraction of either BPA or BPV to KCl, U-46619, histamine, norepinephrine, or serotonin. Thus TNF-alpha appears to selectively inhibit receptor-mediated EDR and NO release in BPA. TNF-alpha-mediated inhibition of EDR differs from that of L-arginine-based inhibitors and may represent an endogenous physiological mechanism of regulation of NO in the endothelium.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1993

Tumor necrosis factor inhibits stimulated but not basal release of nitric oxide.

Tumor necrosis factor alpha (TNF alpha) increases nitric oxide (NO) synthase in vascular endothelium, but it inhibits endothelium-dependent relaxation (EDR) of vascular smooth muscle. We tested whether TNF alpha inhibits the response to, or release of, NO in bovine pulmonary artery (BPA) using the technique of perfusion-superfusion bioassay and ozone chemiluminescence. Effluent from the perfused BPA with endothelium (donor)-relaxed endothelium-rubbed bovine coronary artery (BCA) (detector). Moreover, effluent from the donor stimulated with acetylcholine (ACh) or bradykinin (BK) (0.001 to 100 nmol) relaxed the detector. Direct application of these agonists to the detector failed to produce relaxation. Basal and agonist-stimulated effluent from the donor treated with L-NG-monomethylarginine (LNMMA) (100 microM) suppressed effluent-mediated relaxation of the detector. ACh and BK released LNMMA-inhibitable nitrite and nitrate from the BPA. Thus, the effluent contained NO. Exposure of the donor to TNF alpha (1.25 micrograms/ml) for 60 min did not affect basal release of NO, but it attenuated bioassayable and chemiluminescence-detectable NO release by ACh and BK. The inhibition of NO release was directly related to the magnitude of inhibition of EDR by ACh and BK. Thus, TNF alpha selectively inhibits receptor-mediated release of NO without affecting basal release of NO. This effect differs from that of L-arginine-based inhibitors of NO and represents a unique physiologic mechanism of regulation of NO in the endothelium.

Acetylcholine↗

PubMed1993

Tumor necrosis factor alpha inhibits contractions to sympathetic nerve stimulation by a nitric oxide-dependent mechanism.

Gram-negative sepsis and administration of tumor necrosis factor alpha (TNF alpha) are associated with hypotension and peripheral neuropathies suggestive of impaired sympathetic neurotransmission. We examined the effect of TNF alpha on the responses of the bovine pulmonary artery (BPA) to transmural sympathetic nerve stimulation (SNS). BPA contracted to SNS (0.5-32 Hz, 5-10 V, 2-msec duration, 2-msec delay) in a frequency-dependent manner. The contractions of the BPA to SNS were mediated by norepinephrine and activation of postsynaptic alpha 1-adrenoceptors, since they were attenuated by prazosin. Maximum contraction of the BPA to SNS was significantly enhanced (148 +/- 37% increase, n = 6) after inhibition of nitric oxide synthase with L-NG-monomethylarginine (LNMMA, 500 microM), an effect abrogated by L-arginine (1 mM). TNF alpha (0.0042, 0.042, and 0.42 micrograms/ml) selectively inhibited contractions of the BPA to SNS without affecting the contraction of the BPA to exogenous norepinephrine. In BPA incubated with LNMMA (5-500 microM), TNF alpha facilitated rather than inhibited SNS. TNF alpha increased the formation of amperiometrically measured free nitric oxide in bovine adrenal chromaffin cells in primary culture. The data show that in the absence of LNMMA, TNF alpha releases free nitric oxide from a sympathetic neuron and selectively inhibits the contractions of the BPA to SNS. In BPA in which nitric oxide synthase I is inhibited by LNMMA, TNF alpha amplifies the contractions to SNS, even in the absence of endothelium. Thus, TNF alpha can modify vascular smooth muscle tone by affecting SNS. TNF alpha inhibits SNS at the level of the neuron by a mechanism involving the L-arginine-nitric oxide pathway. TNF alpha-induced suppression of SNS and neurotransmission may contribute to the hypotension and peripheral neuropathy of sepsis.

Acetylcholine↗

PubMedDate not supplied

Ethanol relaxes pulmonary artery by release of prostaglandin and nitric oxide.

Acute-intake of ethanol is associated with vasodilation of vascular smooth muscle (VSM). Relaxation of VSM is dependent, in part, on the actions of nitric oxide (NO) and prostaglandin (PG) produced by endothelial cells (EC) lining the VSM. We examined the effects of endothelium rubbing and inhibition of EC synthesis of NO and PG on ethanol-induced relaxation of bovine pulmonary artery (BPA) and pulmonary vein (BPV) in vitro. Rings of isolated BPA and BPV were mounted in muscle chambers for the isometric recording of force development. Blood vessels were precontracted with an EC50 concentration of the thromboxane receptor mimetic U46619. Ethanol (0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28% (w/v) produced concentration-dependent relaxation of BPA and BPV. Ethanol-induced relaxation was attenuated in BPA with rubbed EC and by the NO synthase inhibitors, L-NG monomethylarginine (LNMMA, 50 microM) and L-nitroarginine (NOLA, 10 microM), and the prostaglandin cyclooxygenase inhibitor, ibuprofen (10 microM). In contrast, ethanol-induced relaxation of BPV was not affected by endothelium rubbing or by NOLA or LNMMA, but was partially attenuated by ibuprofen. Nitric oxide was measured with the chemiluminescence technique. Ethanol increased the content of NO released under basal conditions by the BPA but did not effect basal NO release from BPV. However, ethanol enhanced bradykinin-induced release of NO from BPA and BPV and, at low concentrations, augmented bradykinin-induced relaxation of both BPA and BPV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Ethanol suppresses LPS-induced mRNA for nitric oxide synthase II in alveolar macrophages in vivo and in vitro.

Alcohol abuse increases the incidence and severity of opportunistic lung infections and pneumonias. Inducible nitric oxide (NO) synthase (iNOS II) and NO may be a pivotal system in the intracellular bactericidal activity of macrophages. We tested the hypothesis that acute administration of ethanol (ETOH) suppressed Escherichia coli endotoxin lipopolysaccharide (LPS) mediated upregulation of the iNOS II system in the lung of the rat, in vivo. We also tested the effect of ETOH on alveolar macrophage (AM) production of free NO using microelectrodes. Male Sprague-Dawley rats were given ETOH (5.5 g/kg, IP) 30 min. before giving intratracheal sterile phosphate buffered saline solution (PBS, 0.5 ml) or LPS (1 mg/kg in a total volume of 0.5 ml PBS). The isolated lungs were subjected to bronchoalveolar lavage (BAL) 3.5 hr. later. Aliquots of the BAL fluid were assayed for tumor necrosis factor alpha TNF alpha and reactive nitrogen intermediates (nitrate and nitrite) (RNI) with chemiluminescence. Aliquots of AM were incubated 1 hr ex vivo for spontaneous production of RNI or frozen and assayed for iNOS II mRNA with competitor exchange reverse transcriptase polymerase chain reaction (cERT-PCR). The lung was homogenized and assayed for RNI. LPS increased BAL fluid TNF alpha and RNI, lung RNI, and the spontaneous production of RNI by AM, ex vivo. These effects were inhibited by in vivo administration of inhibitors of iNOS II. LPS increased iNOS mRNA in AM. This was unaffected by iNOS inhibitors. ETOH suppressed LPS-induced BAL fluid TNF, iNOS mRNA and RNI production by AM and the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗