Biomedical subjects

T M McKenna

Publications and source records attributed to T M McKenna.

At least 19 recordsLinked to original sources

PubMed1998

Overexpression of protein kinase C alpha enhances lipopolysaccharide-induced nitric oxide formation in vascular smooth muscle cells.

Our previous studies showed that lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis in cardiovascular tissues is attenuated by protein kinase C (PKC) inhibitors. In the current study, we identify a specific PKC isotype involved in the LPS signal transduction pathway that leads to NO formation in rat vascular smooth muscle cells (VSMC). VSMC were transfected with a mammalian expression vector containing a full length PKCalpha cDNA insert, and a stable transfectant overexpressing PKCalpha was obtained as evidenced by increased expression of PKCalpha mRNA and protein. In response to 100 ng/ml LPS stimulation, the PKCalpha transfectants showed a 1.8-fold increase in PKC activity at 30 min and a twofold increase in NO production over 24 hr compared with cells transfected with control plasmids. The LPS-stimulated increase in NO synthesis in PKCalpha transfectants was blocked by the specific PKCalpha inhibitor Gö 6976. After 6 hr LPS treatment, PKCalpha-transfected and control cells showed equivalent increases in mRNA and protein for the inducible NO synthase. NO synthase activity of the cell extracts assayed in the presence of excess substrate and cofactors was not significantly different between PKCalpha-transfected and control cells after LPS stimulation. However, mRNA levels for GTP cyclohydrolase I, a key enzyme in (6R)-tetrahydro-L-biopterin synthesis, and cationic amino acid transporter-2, involved in L-arginine transport, was enhanced in cells overexpressing PKCalpha compared with control cells. These results suggest that PKCalpha plays an important role in LPS-induced NO formation and that a significant portion of this effect may be by means of enhanced substrate availability to the inducible nitric oxide synthase enzyme.

Amino Acid Transport Systems, Basic↗

PubMed1997

Lipopolysaccharide-responsive protein kinase C isotypes in the adult rat aorta.

Previous studies have shown that protein kinase C (PKC) activity increases in cardiovascular tissue exposed to lipopolysaccharide (LPS). The objective of these experiments was to identify the PKC isotypes that respond to LPS treatment in the adult rat aorta. We found that PKC alpha, -delta, -epsilon, and -zeta isotypes are present in endothelium-intact aortas. The PKC alpha and -epsilon isotypes show two- to threefold increases in abundance after 3 h treatment with 100 ng/mL LPS, while PKC delta and -zeta levels do not increase. In contrast, mRNA for all of the PKC isotypes increased 3.5 to 12-fold during LPS treatment. Both PKC isotype and mRNA levels gradually diminished during 20 h of continuous LPS exposure. Concurrent treatment of the vessels with LPS plus 50 microM cycloheximide caused PKC alpha, -epsilon, and -zeta, but not -delta, isotypes to rapidly decrease in abundance while blunting the increase in PKC isotype mRNA. The major source for all of the PKC isotypes in the vessel is the vascular smooth muscle cells. These results indicate that LPS treatment induces time-dependent increases in PKC isotype mRNA expression and isotype-specific PKC activation and synthesis in vascular tissue.

Age Factors↗

PubMed1997

Vascular smooth muscle cells on Matrigel as a model for LPS-induced hypocontractility and NO formation.

Treatment of vascular tissue with low levels of lipopolysaccharide (LPS) induces nitric oxide synthase (NOS) activity and diminishes vascular contractility. However, in cultured vascular smooth muscle cells (VSMC), very high doses of LPS or the combination of LPS with cytokines are required for the induction of nitric oxide (NO) formation. The aims of this study were to establish a cell model to investigate LPS-induced hypocontractility and NO production and to test the hypothesis that responses of VSMC to LPS are differentiation regulated. We used Matrigel basement membrane matrix to maintain VSMC differentiation and found that VSMC cultured on Matrigel retained significant contractility in response to KCl stimulation. Incubation of VSMC with low levels of LPS(1-100 ng/ml) induced NOS mRNA and protein, induced NO production, and decreased cell contractility in a time- and dose-dependent fashion. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) partially restored LPS-treated VSMC contractility, whereas L-arginine reversed the contractility-restoring effect of L-NAME. These results suggest that VSMC grown on Matrigel are a useful experimental model for investigations into signal transduction mechanisms responsible for LPS-induced vascular hypocontractility.

Animals↗

PubMed1996

Role of nitric oxide in sepsis-induced hyporeactivity in isolated rat lungs.

The aim of the present study was to test the hypothesis that pulmonary microvascular reactivity is depressed in sepsis and that inducible nitric oxide synthase (iNOS) contributes to the vascular hyporeactivity. Rats were made septic by cecal ligation and puncture. After 16 h, pulmonary vascular reactivity was evaluated by measurement of perfusion pressures while the vasculature was challenged with angiotensin II and KCl. The results showed that vascular reactivity was significantly depressed in lungs from septic rats in comparison to sham-operated controls. Pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) restored the depressed vasoreactivity while the nitric oxide (NO) synthase substrate L-arginine (1 mM) reversed the contraction-restoring effect of L-NAME. NO production in lungs from septic rats increased about 4-fold in comparison to sham-operated controls. iNOS protein was expressed in lung tissues, mainly the resistance vessels, from septic rats but not from sham-operated controls. Reverse transcription and polymerase chain reaction also showed a strong induction of iNOS mRNA in lung tissues from septic rats. These results suggest that increased iNOS expression and NO production may contribute to depressed pulmonary vascular reactivity in sepsis.

Animals↗

PubMed1995

PKC mediates LPS- and phorbol-induced cardiac cell nitric oxide synthase activity and hypocontractility.

Lipopolysaccharide (LPS) treatment impairs cardiac myocyte contractility in a nitric oxide synthase (NOS)-dependent manner. The objective of this study was to assess whether protein kinase C (PKC) transduces the LPS signal into an enhanced NOS activity in rat cardiac myocytes. LPS (100 ng/ml) stimulated myocyte PKC activity, inducible NOS (iNOS) expression, and NOS activity in a time- and protein synthesis-dependent fashion. Directly activating PKC with beta-phorbol 12,13-dibutyrate (beta-PDB) also induced myocyte iNOS synthesis and NOS activity and reduced electrically stimulated contractility, while the inactive alpha-PDB was ineffectual. Contractility could be restored to beta-PDB-incubated cells by superfusion with the NOS inhibitor N omega-nitro-L-arginine methyl ester. PKC blockade with sphingosine, chelerythrine, or calphostin-C precluded LPS- and beta-PDB-induced increases in NOS activity and protected contractility. Depletion of PKC by 18 h of incubation with beta-PDB in the presence of chelerythrine also blocked acquisition of enhanced NOS activity and contractile dysfunction when the myocytes were subsequently exposed to LPS. These findings suggest that PKC is a significant intracellular mediator for the effects of LPS on cardiac cell NOS activity and contractile function.

Animals↗

PubMed1994

The brain as a dynamic physical system.

The brain is a dynamic system that is non-linear at multiple levels of analysis. Characterization of its non-linear dynamics is fundamental to our understanding of brain function. Identifying families of attractors in phase space analysis, an approach which has proven valuable in describing non-linear mechanical and electrical systems, can prove valuable in describing a range of behaviors and associated neural activity including sensory and motor repertoires. Additionally, transitions between attractors may serve as useful descriptors for analysing state changes in neurons and neural ensembles. Recent observations of synchronous neural activity, and the emerging capability to record the spatiotemporal dynamics of neural activity by voltage-sensitive dyes and electrode arrays, provide opportunities for observing the population dynamics of neural ensembles within a dynamic systems context. New developments in the experimental physics of complex systems, such as the control of chaotic systems, selection of attractors, attractor switching and transient states, can be a source of powerful new analytical tools and insights into the dynamics of neural systems.

Action Potentials↗

PubMed1994

Protein kinase C is a mediator of lipopolysaccharide-induced vascular suppression in the rat aorta.

Treatment of vascular tissue with lipopolysaccharide (LPS) in vitro induces hyporesponsiveness to contractile agonists. We investigated whether protein kinase C (PKC) transduces the LPS signal into contractile dysfunction. Rat aortic tissue was incubated .5-18 h with LPS (10 or 30 ng/mL) or alpha- and beta-phorbol 12,13-dibutyrate (PDB, .1 or 1 microM), either alone or combined with cycloheximide (50 microM) or the kinase inhibitors sphingosine (20 microM), H7 (1-(5-isoquinolinylsulfonyl)-2-methyl piperazine, 25 microM), and HA1004 (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, 25 microM). LPS and beta-PDB induced a sustained translocation of PKC activity from the cytosol to the membrane, an increased protein synthesis-dependent expression of nitric oxide synthase (NOS) activity, and an impaired contractility that could be partially reversed by treatment with the NOS inhibitor N omega-nitro-L-arginine methyl ester. Incubation with alpha-PDB, an inactive isomer of beta-PDB, did not alter any of the tissue functions. Sphingosine blocked LPS- and beta-PDB-induced NOS activity and LPS-induced impairments in tissue contractility and PKC translocation. Incubation with H7 also protected against LPS-induced vasoplegia, while HA1004, used as a negative control for H7, provided little protection against LPS. These data indicate that PKC plays a role as an intracellular mediator of LPS-induced NOS activity and vascular suppression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

In vitro endotoxin exposure induces contractile dysfunction in adult rat cardiac myocytes.

In vivo endotoxin treatment causes a nitric oxide-mediated hypocontractility in cardiac myocytes. The objective of this study was to assess whether in vitro endotoxin exposure confers similar contractile defects in adult rat cardiac cells. We found that incubation of cardiac myocytes for 6 h with 10-100 ng/ml endotoxin resulted in progressive time- and protein synthesis-dependent decreases in electrically stimulated twitch magnitudes and increased contraction and relaxation times. Serum was not required for the endotoxin-induced hypocontractility. The endotoxin-induced defect in contractility was reversed over time, since myocytes continuously incubated with endotoxin for 24 h exhibited normal contractility; in contrast, control cells incubated for 18 h were suppressed by a subsequent 6-h exposure to endotoxin. Nitric oxide synthase activity was increased after a 6-h endotoxin treatment as evidenced by a dose-dependent enhanced conversion of [3H]arginine to [3H]citrulline and by elevated guanosine 3',5'-cyclic monophosphate levels. Superfusion of endotoxin-incubated cells with N omega-nitro-L-arginine methyl ester restored contractile function, whereas superfusion with L-arginine reimposed abnormal contractility. Naive myocytes superfused with 8-bromoguanosine 3',5'-cyclic monophosphate expressed contractile defects similar to those induced by endotoxin. These findings demonstrate that endotoxin has direct negative effects on cardiac cell contractile function and that induction of NO synthase activity is a primary intracellular mediator of the diminished contractility.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Lipopolysaccharide detoxification by endotoxin neutralizing protein.

Endotoxin neutralizing protein (ENP), a recombinant form of the anti-lipopolysaccharide factor that was isolated from amebocytes of the American horseshoe crab, Limulus polyphemus, detoxifies lipopolysaccharide (LPS) both in vitro and in vivo. Using the Limulus amebocyte lysate assay, LPS was detoxified by ENP at a 1 to 1 weight ratio (1:1). When isolated rat aortic rings were preincubated for 16 hr with either LPS or LPS/ENP (1:5), only aortas in the LPS/ENP group contracted normally under norepinephrine stimulation. To show that detoxification of a lethal amount of LPS (18 mg/kg, LD50 at 48 hr) persists in vivo, LPS/albumin (1:1) or LPS/ENP (1:1) mixtures were preincubated (30 min, 37 degrees C) and then injected intravenously into rats. In the 8 hr after injection, LPS/ENP challenged rats, in contrast to their LPS/albumin injected counterparts, had significantly fewer physical signs of acute LPS toxicity (P < 0.001). At 48 hr after challenge, all LPS/ENP treated rats survived (P < 0.01 vs LPS/albumin), and with significantly less weight loss (P < 0.001 vs LPS/albumin challenged survivors). At necropsy, the LPS/ENP group was free of typical LPS-induced gross organ lesions, notably in the liver, spleen, gut-associated lymphoid tissue (GALT), and small intestine. By microscopic examination, lymphocytic necrosis in the spleen and GALT of the LPS/ENP treated survivors was significantly milder than that in the LPS/albumin challenged survivors, although the degree of hepatocellular necrosis and small intestinal enteritis was similar. LPS-neutralizing proteins such as ENP may be useful in treating LPS toxicity.

Amino Acid Sequence↗

PubMed1992

Recovery of vascular tissue contractile function during sustained endotoxin exposure.

Rat aortic rings incubated in vitro with endotoxin (10 ng/ml, 24 h) exhibited marked suppressions of sensitivity and maximum contraction to norepinephrine as well as a significant increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels. Increasing the incubation time to 42 h did not result in greater suppression; instead, these rings showed improved contractile performance and control-level cGMP. Rings incubated with endotoxin for 18 h and then allowed to recuperate in control medium for another 24 h exhibited normal maximal contractions and cGMP levels; however, these rings remained less sensitive to norepinephrine. An endotoxin-neutralizing protein (50 ng/ml) shielded rings from endotoxin-induced contractile suppression if present during incubation but was unable to restore tissue sensitivity to normal levels if present during the 24-h recuperation. These findings show that vascular tissue exposed to endotoxin has a varied and time-dependent ability to counter the effects of endotoxin on contractile function and intracellular cGMP.

Animals↗

PubMed1992

In vivo and in vitro effects of endotoxin on vascular responsiveness to norepinephrine and signal transduction in the rat.

We investigated, after in vitro and in vivo exposure to gram-negative endotoxin, the altered responsiveness of rat aortic smooth muscle to catecholamines. Two hour exposure of aortic rings from normal rats to 100 ng/ml of Escherichia coli 0111:B4 endotoxin in vitro in an artificial medium supplemented with 5% fetal calf serum at 37 degrees C did not effect the basal and norepinephrine (NE)-stimulated (10 microM, 1 hr, 37 degrees C) phosphoinositide (PI) hydrolysis and isometric contractions induced by graded doses (1 nM to 10.0 microM) of NE. Increasing the incubation time with endotoxin to 18 hr did not alter the basal PI hydrolysis but significantly (P less than 0.05) decreased the NE-induced PI hydrolysis (30% inhibition) and contractile sensitivity to NE (increase of EC50 from 20.0 +/- 3.8 to 156.4 +/- 46.7 nM). Qualitatively similar results were obtained in experiments where rats were injected intravenously with buffer or an LD50 dose (10 mg/kg) of endotoxin. In these ex vivo measurements, only an 18 hr exposure to endotoxin caused significant (P less than 0.001) decreases in basal (58% inhibition) and NE-stimulated (75% inhibition) PI hydrolysis and in NE-induced isometric contractions (increase of EC50 from 11.0 +/- 3.3 to 664.1 +/- 280.0 nM). The results show that the endotoxin-induced hyporeactivity to alpha 1-adrenergic receptor stimulation 1) is markedly dependent on the length of endotoxin exposure, 2) does not require (although may be enhanced by) contact with blood cells and plasma, and 3) is paralleled by a decrease in both basal and NE-stimulated PI hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Pentoxifylline treatment of sepsis in conscious Yucatan minipigs.

Recent evidence suggests that pentoxifylline (PTX) may be useful in the treatment of sepsis. We examined effects of PTX in a conscious swine model of sepsis. Yucatan minipigs (20-30 kg) were anesthetized and instrumented with catheters in the vena cava, aortic arch, pulmonary artery (Swan-Ganz thermodilution catheter), and peritoneum. Twenty-four hours after surgery, sepsis was induced by intraperitoneal (ip) injection of Escherichia coli bacteria (2 x 10(10) cfu/kg). Nonseptic pigs received intraperitoneal saline (5 ml/kg). PTX treatment (3 mg/kg/hr, iv; 1 mg/ml in 0.9% saline) and maintenance fluid (5 ml/kg/hr, iv) were started with bacterial infusion. An additional 60 cc/kg 0.9% saline bolus was administered iv at 1 hr. Pigs were monitored before and 1, 2, 5, and 24 hr after bacterial injection. Intraperitoneal injection of bacteria led to significant reductions in blood pressure and cardiac output and elevations in pulmonary wedge pressure and pulmonary vascular resistance. These effects were attenuated by PTX treatment. All septic animals demonstrated elevated creatinine, blood urea nitrogen, circulating endotoxin (LPS), and tumor necrosis factor concentrations, reductions in white blood cell and platelet counts, and peritonitis. None of these responses was altered by PTX treatment. We conclude that PTX may prove to be a useful therapeutic tool in the early treatment of septic shock but is limited in the scope of its effects.

Animals↗

PubMed1992

Differential contractile responses of mesenteric and pulmonary artery segments to norepinephrine and phorbol ester in the septic pig.

The contractile response of isolated vascular segments was studied in Yucatan miniature swine approximately 48 hr after induction of sepsis by intraperitoneal injection of live Escherichia coli. Compared to non-septic controls, segments of the cranial mesenteric artery from septic animals showed a significantly attenuated contractile response to the adrenergic receptor agonist norepinephrine (NE). The EC50 for NE increased from 1.1 +/- 0.3 to 6.3 +/- 2.0 microM and the Emax decreased from 1,010 +/- 179 to 387 +/- 75 mg tension/mg tissue. In contrast, segments of the pulmonary artery showed no significant difference in contractility to NE between sham-operated and septic animals. Mesenteric and pulmonary artery segments from both septic and control animals exhibited similar contraction to the protein kinase C activator phorbol-12, 13-dibutyrate. This suggests that the observed hyporeactivity to NE in porcine mesenteric artery segments is not simply due to cellular damage by toxins associated with the septic state. The results also indicate that the impact of gram-negative sepsis on vascular contractile function varies between tissue from the systemic and pulmonary circulation in pigs.

Animals↗

PubMed1992

Effects of in vivo pentoxifylline treatment on survival and ex vivo vascular contractility in a rat lipopolysaccharide shock model.

Depending on the dose and dosing, pentoxifylline (PTX) treatment can improve or worsen survival from lipopolysaccharide (LPS) shock in rats. Intraperitoneal (i.p.) PTX, 20 mg/kg, administered once 15 min after intravenous (i.v.) LPS (17 mg/kg), significantly improved survival in unanesthetized LPS-shocked rats. Multiple 20 mg/kg PTX injections (five total, spaced at 45 min intervals starting 15 min after LPS) significantly worsened survival. A lower dose, 12 mg/kg, given as a single or multiple injections, did not alter survival. We tested the ex vivo contractile response to norepinephrine (NE) of aortic rings isolated 3.75 hr after i.v. injection of PBS or LPS. Both untreated LPS-shocked and multiple 12 mg/kg PTX treated normal rats (i.v. PBS) had significantly diminished maximum contractility. The ex vivo vascular hypocontractility found in untreated LPS-shocked rats was not aggravated nor ameliorated by multiple 12 mg/kg PTX injections. The ex vivo effects on contractility of multiple 20 mg/kg PTX treatment of LPS shock could not be studied because survival times were shorter than 3.5 hr. In using PTX to treat LPS shock, potentially harmful vasodilation must be considered.

Animals↗

PubMed1990

Prolonged exposure of rat aorta to low levels of endotoxin in vitro results in impaired contractility. Association with vascular cytokine release.

Treatment of volunteers or animals with endotoxin in vivo results in reduced vascular reactivity to catecholamines. Endotoxin also causes liberation of the vasoactive cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) from vascular smooth muscle and endothelial cells in culture. This study tested whether defects in contractility could be induced in isolated vascular tissue by prolonged exposure to endotoxin (1-100 ng/ml) in vitro, and whether IL-1 and TNF release by blood vessels is altered during the establishment of endotoxin induced contractile dysfunction. A concentration of endotoxin as low as 1 ng/ml suppressed contractions to norepinephrine (NE) and KCl; aortic sensitivity to NE also decreased. The presence of serum constituents or an intact endothelium were not necessary for endotoxin-induced vascular suppression. Aortas incubated with endotoxin liberated IL-1 and TNF in a dose-dependent fashion. The addition of dexamethasone or indomethacin during the incubations generally suppressed release of the cytokines and improved tissue reactivity to NE. The endotoxin-induced diminished vascular contraction and augmented IL-1 and TNF liberation required de novo protein synthesis; tissue incubated with endotoxin plus actinomycin D was completely shielded from the influence of endotoxin on vascular reactivity to NE. The association between endotoxin-induced vascular cytokine release and diminished contraction suggests a possible role for cytokines derived from the vasculature in the regulation of contractile function.

Animals↗

PubMed1989

Responses of single auditory cortical neurons to tone sequences.

The responses of single neurons in the primary and secondary auditory cortex of cat were recorded during the presentation of sequences consisting of five tones of different frequencies. Discharges to tones within these sequences usually (84%) exhibited a dependence on the 'direction' of the sequence (ascending, descending, or mixed frequencies). For sequences consisting of 5 tones of identical frequency (monotone) the response often depended on serial position, including cases in which the neuron only responded to later tones in the sequence. Comparison of responses to heterogeneous and monotone sequences showed that response dependence on serial position was a factor in response dependence on sequence direction. Auditory cortical neurons can exhibit stronger responses to a tone presented in a sequence than to the same tone presented alone. Hence, the responses to tones within sequences may not be highly predictable from the responses to isolated tones.

Acoustic Stimulation↗

PubMed1989

Cholinergic modulation of frequency receptive fields in auditory cortex: I. Frequency-specific effects of muscarinic agonists.

Previously we reported that acetylcholine (ACh) and acetyl-beta-methacholine (MCh) modify responses of neurons in auditory cortex to individual frequencies. The purpose of this study was to determine whether muscarinic agonists produce frequency-specific alterations or general changes in cellular responses. Frequency-specific modifications would be evident in alterations of frequency receptive fields (FRF) that differed across frequencies while general effects would be seen as changes that were more or less the same over frequencies. Responses of single neurons to designated sets of tones were recorded in the auditory cortex of chronically prepared awake cats before, during, and following ejection of ACh or MCh by iontophoresis or micropressure using multibarrel micropipettes. Frequency receptive fields were determined by presenting isointensity tones across a range of frequencies including the cell's best frequency (BF) to tone onset. FRF for "off" and "sustained (through)" responses were also determined quantitatively. The effects of ACh and MCh were predominantly frequency-specific (77%, 39/51 cells); general changes (19%, 10/51) and no effects (4%, 2/51) were less likely. Frequency-specific effects involved both facilitation and reduction of the same response component to different frequencies within the same neuron. For responses to tone onset (but not "through" and "off" responses), agonists were more likely to produce a decrease at the BF while simultaneously increasing responses to other frequencies. Agonists could increase or decrease frequency selectivity. Effects of agonists could be blocked by atropine, suggesting involvement of muscarinic receptors.

Acetylcholine↗

PubMed1989

Cholinergic modulation of frequency receptive fields in auditory cortex: II. Frequency-specific effects of anticholinesterases provide evidence for a modulatory action of endogenous ACh.

Exogenously applied muscarinic agonists--for example, acetylcholine (ACh) and acetyl-beta-methacholine (MCh)--modify frequency receptive fields in auditory cortex of unanesthetized animals in a frequency-specific rather than global manner. The present study sought to relate these findings to endogenous actions of ACh by using the anticholinesterase agents eserine sulphate and soman (0-1,2,2-trimethylpropylmethylphosphonofluoridate) to facilitate the effects of endogenous ACh. Frequency receptive fields (FRF) were determined by presenting sequences of different isointensity tones before, during, and after application of ACh, MCh, eserine, or soman; also the cholinesterase blockers were applied between applications of ACh or MCh. The major effects produced by the inhibitors were similar to those of the agonists. Predominant effects were frequency-specific changes in FRF. Further, eserine and soman, similar to ACh and MCh, produced shifts in the best frequency (BF) of FRF due mainly to coordinated depression of responses to the BF and increased responses to adjacent, non-BF. The results indicate that exogenous and endogenous ACh, acting via muscarinic receptors, can significantly influence the physiological functioning of cortical neurons and consequently their processing of sensory information.

Acetylcholine↗