Comparison of the pharmacological profile of S-nitrosothiols, nitric oxide and the nitrergic neurotransmitter in the canine ileocolonic junction

1. In organ bath experiments, hydroquinone (30-100 microM) and hydroxocobalamin (30-100 microM) concentration-dependently inhibited the relaxations induced by NO (0.3-30 microM) but not those by nitroglycerin (GTN, 1 microM) in the canine ileocolonic junction (ICJ). Hydroxocobalamin reduced the relaxation to low frequency (2 Hz) stimulation of the non-adrenergic, non-cholinergic (NANC) nerves, whereas hydroquinone only reduced the NANC nerve-mediated relaxations to electrical stimulation at 16 Hz, 0.5 ms. 2. Relaxations to S-nitroso-L-cysteine (CysNO, 1-30 microM), or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1-30 microM) were not inhibited by hydroquinone (30-100 microM), hydroxocobalamin (30-100 microM), pyrogallol (30-100 microM) or L-cysteine (1-3 microM). Hydroquinone (100 microM) only reduced the relaxation to 10 microM CysNO. Hydroxocobalamin, but not hydroquinone, pyrogallol or L-cysteine, potentiated the relaxations to the lowest concentration (1 microM) of S-nitrosoglutathione (GSNO, 1-30 microM). 3. In the superfusion bioassay, hydroquinone (100 microM) and hydroxocobalamin (1 microM) concentration-dependently inhibited the biological activity of authentic NO (1-4 pmol) to the same extent as that of the transferable nitrergic factor, released from the canine ICJ in response to NANC nerve stimulation (8-16 Hz, 2 ms). Responses to GTN (10 pmol) or adenosine 5'-triphosphate (10 nmol) were not affected. 4. In conclusion, the nitrosothiols CysNO, SNAP and GSNO relax the canine ileocolonic junction, but these relaxations, pharmacologically, behave differently from the NANC nerve-mediated relaxations. From the bioassay experiments, we conclude that the nitrergic factor, released in response to NANCnerve stimulation of the canine ICJ, behaves pharmacologically like NO but not like a nitrosothiol.Therefore, we suggest NO, and not CysNO, SNAP or GSNO as the inhibitory NANC neurotransmitter in the canine ICJ.     

The effect of changing excitation frequency on parallel conductance in different sized hearts

OBJECTIVE: An important component of the ventricular volume measured using the conductance catheter technique is due to parallel conductance (Vc), which results from the extension of the electric field beyond the ventricular blood pool. Parallel conductance volume is normally estimated using the saline dilution method (Vc(saline dilution)), in which the conductivity of blood in the ventricle is transiently increased by injection of hypertonic saline. A simpler alternative has been reported by Gawne et al. [12]. Vc(dual frequency) is estimated from the difference in total conductance measured at two exciting frequencies and the method is based on the assumption that parallel conductance is mainly capacitive and hence is negligible at low frequency. The objective of this study was to determine whether the dual frequency technique could be used to substitute the saline dilution method to estimate Vc in different sized hearts. METHODS: The accuracy and linearity of a custom-built conductance catheter (CC) system was initially assessed in vitro. Subsequently, a CC and micromanometer were inserted into the left ventricle of seven 5 kg pigs (group 1) and six 50 kg pigs (group 2). Cardiac output was determined using thermodilution (group 1) and an ultrasonic flow probe (group 2) from which the slope coefficient (alpha) was determined. Steady state measurements and Vc estimated using saline dilution were performed at frequencies in the range of 5-40 kHz. All measurements were made at end-expiration. Finally, Vc was estimated from the change in end-systolic conductance between 5 kHz and 40 kHz using the dual frequency technique of Gawne et al. [12]. RESULTS: There was no change in measured volume of a simple insulated cylindrical model when the stimulating frequency was varied from 5-40 kHz. Vc(saline dilution) varied significantly with frequency in group 1 (8.63 +/- 2.74 ml at 5 kHz; 11.51 +/- 2.65 ml at 40 kHz) (p = 0.01). Similar results were obtained in group 2 (69.43 +/- 27.76 ml at 5 kHz; 101.24 +/- 15.21 ml at 40 kHz) (p < 0.001). However, the data indicate that the resistive component of the parallel conductance is substantial (Vc at 0 Hz estimated as 8.01 ml in group 1 and 62.3 ml in group 2). There was an increase in alpha with frequency in both groups but this did not reach significance. The correspondence between Vc(dual frequency) and Vc(saline dilution) methods was poor (group 1 R2 = 0.69; group 2 R2 = 0.22). CONCLUSION: At a lower excitation frequency of 5 kHz a smaller percentage of the electric current extends beyond the blood pool so parallel conductance is reduced. While parallel conductance is frequency dependent, it has a substantial resistive component. The dual frequency method is based on the assumption that parallel conductance is negligible at low frequencies and this is clearly not the case. The results of this study confirm that the dual frequency technique cannot be used to substitute the saline dilution technique.     

Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious, hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3-4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 microg kg(-1) h(-1), i.v.) 2. The early (1-2 h) hypotension in SD rats (-11+/-3 mmHg; n=7) was significantly less than that in TG rats (-35+/-3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108+/-3 mmHg; 24 h LPS, 112+/-4 mmHg; TG rats: baseline, 171+/-2 mmHg; 24 h LPS, 169+/-3 mmHg). At this stage in the SD rats there was a renal vasodilatation (delta vascular conductance, 29+/-10 [kHz mmHg(-1)]10(3)) but not in TG rats (delta vascular conductance 2+/-3[kHz mmHg(-1)]10(3)). 3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 microg kg(-1) bolus, 600 microg kg(-1) h(-1)) between 24 and 31 h in SD rats caused a fall in MAP of 16+/-2 mmHg accompanied by hindquarters vasodilatation (delta vascular conductance 11+/-3 (kHz mmHg(-1))10(3)). In TG rats, under the same conditions, the fall in MAP was -60+/-6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (delta vascular conductance, 23+/-5, 32+/-7, and 14+/-4 (kHz mmHg(-1))10(3), respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats. 4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was -17+/-4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5+/-3, -4+/-5, and 12+/-4 (kHz mmHg(-1)10(3), respectively. 5. Administration of the angiotensin (AT1)-receptor antagonist, losartan (10 mg kg(-1), i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (-12+/-3 and -14+/-4 mmHg, respectively). 6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.     

Reduced inward rectifying and increased E-4031-sensitive K+ current density in arrhythmogenic subendocardial purkinje myocytes from the infarcted heart

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.     

Zoonoses and potential zoonoses transmitted by bats

PURPOSE: The purpose of this study was to develop an analytical method for the quantitative determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF using Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFT) with Kubelka-Munk function analysis. METHODS: Carbopol 974P NF is a high molecular weight, chemically crosslinked polymer of acrylic acid, that has the C=O stretching band of the unionized carboxylic acid function at 1695 cm(-1). The quantitative determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF is based upon the asymmetrical C=O stretching of the carboxylate anion at 1570 cm(-1) measured by DRIFT spectroscopy. RESULTS: To overcome spectral differences arising from sample preparation (powders, granules and tablets) and in an effort to increase the precision of the analytical method, the following approaches were used: (1) an internal standard, (2) first derivative of the spectrum to eliminate the effect of baseline drift and (3) the ratio of the first derivative of the C=O stretch of the carboxylate anion peak (1570 cm(-1)) in the neutralized Carbopol 974P NF to that of the peak of the internal standard (866 cm(-1)). The above data treatment techniques proved to be superior to the usual methods of peak height or peak area. The calibration curve of the ratio of the first derivative (1570 cm(-1)/866 cm(-1)) was a linear function of the mass of sodium carboxylate over the range from 0.0% to 100.0% neutralization of the carboxylic acid function in Carbopol 974P NF (Fig. 1a). No particle size or sample preparation effects were noted within the experimental error. CONCLUSIONS: DRIFT spectroscopy using the Kubelka-Munk function is a powerful tool for the routine determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF in complex pharmaceutical formulations.     

Pulmonary vasodilator response to adenosine triphosphate-sensitive potassium channel activation is attenuated during desflurane but preserved during sevoflurane anesthesia compared with the conscious state

BACKGROUND: The objective of this study was to investigate the effects of sevoflurane and desflurane anesthesia on the pulmonary vasodilator response to the adenosine triphosphate-sensitive potassium channel agonist, lemakalim, compared with the response measured in the conscious state. In addition, the authors assessed the extent to which sympathetic alpha1-adrenoreceptor inhibition and cyclooxygenase pathway inhibition modulate the vasodilator response to lemakalim. METHODS: Twenty-four conditioned male mongrel dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow relationship. After preconstriction with the thromboxane analogue, U46619, dose-response relationships to lemakalim were assessed on separate days in the conscious state and during sevoflurane (approximately 3.5% end-tidal) and desflurane (approximately 10.5% end-tidal) anesthesia (approximately 1.5 minimum alveolar concentration for each anesthetic agent). The effects of sympathetic alpha1-adrenoreceptor inhibition (prazosin) and cyclooxygenase inhibition (indomethacin) on the pulmonary vasodilator response to lemakalim also were assessed in the conscious and desflurane-anesthetized states. RESULTS: Neither sevoflurane nor desflurane had a net effect on the baseline left pulmonary vascular pressure-flow relationship compared with the conscious state. The magnitude of the pulmonary vasodilator response to lemakalim was preserved during sevoflurane anesthesia but was attenuated (P < 0.05) during desflurane anesthesia compared with the conscious state. The attenuated lemakalim-induced vasodilator response during desflurane anesthesia was partially reversed (P < 0.05) by pretreatment with prazosin but not indomethacin. CONCLUSION: These results indicate that adenosine triphosphate-sensitive potassium channel-mediated pulmonary vasodilation is preserved during sevoflurane anesthesia but is attenuated during desflurane anesthesia. The attenuated response to adenosine triphosphate-sensitive potassium channel activation during desflurane anesthesia is partially mediated by reflex sympathetic alpha1-adrenoreceptor vasoconstriction.     

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.     

The influence of antibodies to TNF-alpha and IL-1beta on haemodynamic responses to the cytokines, and to lipopolysaccharide, in conscious rats

Male, Long Evans rats (350-450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-alpha and human recombinant interleukin-1 (IL-1)beta, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-alpha and IL-1beta on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-alpha and IL-1beta had any influence on the responses to lipopolysaccharide (LPS). TNF-alpha (10, 100 and 250 microg kg(-1), in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum delta, +101+/-9 beats min(-1)) and modest hypotension (maximum delta, -10+/-2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum delta, +6+/-6, +27+/-9, and +61+/-12% at 10, 100 and 250 microg kg(-1), respectively). IL-1beta (1, 10, and 100 microg kg(-1) in separate groups, n = 8, 8 and 9, respectively) evoked changes similar to those of TNF-alpha (maximum delta heart rate, +69+/-15 beats min(-1); maximum delta mean blood pressure, -14+/-2 mmHg; maximum delta hindquarters vascular conductance, +49+/-17%), but with no clear dose-dependency. TNF-alpha (250 microg kg(-1)) and IL-1beta (10 microg kg(-1)) together caused tachycardia (maximum delta, +76+/-15 beats min(-1)) and hypotension (maximum A, -24+/-2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52+/-6%, +23+/-8%, and +52+/-11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum delta, -50+/-3% and -58+/-3%, respectively). Although bolus injection of LPS (3.5 mg kg(-1)) caused an initial hypotension (maximum delta, -27+/-11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-alpha and IL-1beta or after bolus injection of LPS, the secondary reduction in blood pressure was similar (-16+/-2 and -13+/-3 mmHg, respectively), but in the former group the tachycardia (+117+/-14 beats min(-1)) and increase in hindquarters vascular conductance (+99+/-21%) were greater than after bolus injection of LPS (+54+/-16 beats min ' and +439%, respectively). Pretreatment with antibodies to TNF-alpha and IL-1beta (300 mg kg(-1)) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h. Pretreatment with antibodies to TNF-alpha and IL-1beta before infusion of LPS (150 microg kg(-1) h(-1) for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion. the results indicate that although co-administration of TNF-alpha and IL-1beta can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.