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We have examined potential functions of nitric oxide (NO) within the paraventricular nucleus (PVN) in urethan-anesthetized male Sprague-Dawley rats. Initial experiments demonstrated microinjection of 50 pmol of the NO donor, sodium nitroprusside (SNP), directly into the PVN resulted in significant decreases in mean blood pressure (BP) (-3,312 +/- 1,189 mmHg/s over 300-s response time; P < 0.05). To determine whether such effects were attributable to SNP-induced NO release, NO was administered into PVN directly by bilateral microdialysis of NO-containing artificial cerebrospinal fluid (NO-aCSF), a process that results in delivery of approximately 50 pmol NO.PVN-1 x min-1. Such microdialysis resulted in significant decreases in BP (-5,121 +/- 817 mmHg/s over 1,200-s response time; P < 0.005), while aCSF microdialysis was without effect (1,298 +/- 1,071 mmHg/s over 1,200-s response time; P > 0.1). Amino acid concentrations were measured in dialysates collected during perfusion of the same PVN sites with either aCSF or NO-aCSF by high-performance liquid chromatography (HPLC) analysis. NO-aCSF induced significant increases in aspartate (aCSF 31 +/- 7 pmol/30 min; NO-aCSF 134 +/- 33 pmol/30 min; P < 0.05), glutamate (aCSF 36 +/- 5 pmol/30 min; NO-aCSF 417 +/- 108 pmol/30 min; P < 0.02), gamma-aminobutyric acid (aCSF 4.1 +/- 0.7 pmol/30 min; NO-aCSF 104 +/- 29 pmol/30 min; P < 0.02), and taurine (aCSF 34 +/- 3 pmol/30 min; NO-aCSF 117 +/- 24 pmol/30 min; P < 0.01) concentrations, while alanine, glutamine, and serine concentrations were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat aorta was homogenized and the 13000 x g supernatant fraction was tested for heme oxygenase (HO) activity by using a sensitive gas chromatographic method to measure carbon monoxide (CO), one of the products of the HO reaction. The rate of NADPH-dependent CO formation, an index of HO activity, was 1.41 +/- 0.40 nmol CO.mg(-1)protein. h(-1) in the rat aorta supernatant fraction and 2.05 +/- 0.55 nmol CO.mg(-1) protein.h(-1) in the rat liver 13000 x g supernatant fraction, a tissue known to contain HO activity. Chromium mesoporphyrin (0.05 mM), an inhibitor of rat liver HO, significantly decreased HO activity by 26% in the aorta supernatant fraction and 50% in the liver supernatant fraction. On the basis of the results of this study, which demonstrated HO-catalyzed CO formation in aortic tissue, and previous observations that CO relaxes vascular smooth muscle, we suggest that a physiological role for CO in vascular smooth muscle relaxation should be further investigated.

1 We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction controlling the formation of endothelin-1 (ET-1). In the present study, we have examined whether CO is formed naturally in the vessel. 2 The CO-forming enzyme, haem oxygenase (HO), was identi®ed in ductal tissue in its constitutive (HO-2) and inducible (HO-1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO-1 was localized to endothelial and muscle cells, while HO-2 was found only in muscle cells. Inside the muscle cells, HO-1 and HO-2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO-1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3 CO was formed enzymatically from added substrate (hemin, 50 mM) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 mM). 4 ZnPP (10 mM) had no e ect on the tone of the ductus under normal conditions (2.5 to 95% O 2 ), but it contracted the endotoxin-treated ductus (at 2.5% O 2 ). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O 2 ). 5 ZnPP (10 mM) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 mM)-contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6 We conclude that CO is formed in the ductus and may exert a relaxing in¯uence when its synthesis is upregulated by an appropriate stimulus.

We recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2-keto-deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb Pstl-BamHl fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5 K, 5.1 K and 5.5 K. Only the 5.5 K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl-BamHl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.

Endogenous carbon monoxide (CO) formation has been measured in different biological systems using a variety of analytical procedures. The methods include gas chromatography-reduction gas detection, gas chromatography-mass spectroscopic detection, laser sensor-infrared absorption, UV-visible spectrophotometric measurement of CO-hemoglobin or CO-myoglobin complex, and formation of (14)CO from (14)C-heme formed following [2-(14)C]glycine administration. CO formation ranged from a low of 0.029 nmol/mg of protein/h in chorionic villi of term human placenta to a high of 0.28 nmol/mg of protein/h in rat olfactory receptor neurons in culture and rat liver perfusate.

No abstract

Chronic experiments were done on six dogs fitted with EMG electrodes on pharyngeal and esophageal musculature. Electromyographic activity of the cricopharyngeus was recorded in awake and sedated animals with and without manometric recordings as well as during esophageal distension. Intraluminal upper esophageal sphincter (UES) pressure had two distinct components; active contraction accompanied by cricopharyngeal EMG activity and passive elasticity that persisted in the absence of EMG activity. Between swallows, the cricopharyngeal EMG activity patterns observed were of either tonic activity, no activity, or phasic activity with inspiratory bursts. The activity level was markedly affected by anesthesia, phonating, whining, panting, level of alertness, or changes in head posture. A brisk UES contraction was elicited in response to passage of the manometric assembly and to intraesophageal balloon distension. Persistent EMG augmentation after stationing of the manometric sensor suggested that intraluminal manometry tends to exaggerate resting sphincter pressure. We conclude that electrical activity of the cricopharyngeus, and by inference UES pressure, is markedly affected by many variables that are difficult to control during clinical or experimental determinations of UES pressure.