Charles S. Bockman

Isolation, structural characterization and pharmacological activity of dog neuromedin U

The neuromedin U-like immunoreactivity in an extract of dog small intestine was resolved by reversed-phase HPLC into two molecular forms. The primary structure of the larger form (NMU-25) was established as: Phe-Arg-Leu-Asp-Glu-Glu-Phe-Gln-Gly-Pro10-Ile-Ala-Ser-Gln-Val-Arg- Arg-Gln-Phe- Leu20-Phe-Arg-Pro-Arg-Asn-NH2. This sequence shows five substitutions relative to pig neuromedin U-25. The primary structure of the second peptide (NMU-8) was established as: pGlu-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The sequence contains the substitution pGlu for Tyr1 compared with pig neuromedin U-8. The potency of synthetic dog NMU-8 in contracting smooth muscle from the rat uterus (EC50 10 +/- 2 nM; mean +/- S.E., n = 6) was not significantly different from the corresponding potency of pig NMU-8 (EC50 16 +/- 5 nM) but the maximum response produced by the dog peptide was greater (58%; p less than 0.05) than that produced by pig NMU-8.

Enhanced release of endothelium-derived relaxing factor in mineralocorticoid hypertension

Ring segments of superior mesenteric arteries studied in vitro were used to determine the role of the vascular endothelium in regulating vascular contractile and relaxant sensitivity in deoxycorticosterone acetate (DOCA)-salt hypertension. Wistar rats were given DOCA (20 mg/kg s.c. twice per week) and 1% NaCl drinking water for 5 weeks. In ring segments containing endothelium, there was a decrease in contractile sensitivity to arginine vasopressin, no change in contractile sensitivity to norepinephrine and KCl, and no change in relaxant sensitivity to acetylcholine or isoproterenol in arteries from hypertensive rats compared with normotensive controls. Removal of the vascular endothelium by rubbing had no effect on the contractile response to arginine vasopressin and KCl or the relaxant response to isoproterenol in control arteries. In arteries without endothelium, DOCA-salt hypertension caused a threefold increase in contractile sensitivity for arginine vasopressin, norepinephrine, and KCl; a 50% reduction in maximal relaxation to isoproterenol; and a threefold decrease in relaxant sensitivity to sodium nitroprusside. Indomethacin (10 microM) had no effect on contraction or relaxation. However, N-monomethyl L-arginine unmasked altered contractile sensitivity to norepinephrine in arteries from DOCA-salt hypertensive rats. These data show that the endothelium compensates for increased contractile and reduced relaxant responses of vascular muscle in DOCA-salt hypertension by increasing the release of endothelium-derived relaxing factor. These data suggest that altered vascular responsiveness is masked by the endothelium, thus preventing these alterations from contributing to increased peripheral resistance during the development of DOCA-salt hypertension.

Primary structure and pharmacological activity of a nonapeptide related to neuromedin U isolated from chicken intestine.

An extract of chicken intestine contained neuromedin U-like immunoreactivity (36 pmol/g wet tissue weight). The primary structure of the predominant molecular form (NMU-9), comprising 94% of the total immunoreactivity, was established as: Gly-Tyr-Phe-Phe-Phe-Arg-Pro-Arg- Asn-NH2. This sequence differs from that of pig neuromedin U-8 (NMU-8) by the substitution of Leu3 by Phe and, like the corresponding peptide from the guinea pig, is extended from the NH2-terminus by a Gly residue. A minor component of neuromedin U comprised 25 amino acid residues. An extract of chicken whole brain contained much less NMU-like immunoreactivity (1.5 pmol/g) and the nonpeptide was the only molecular form detected. Synthetic chicken NMU-9 produced a concentration-dependent contraction of smooth muscle from the rat uterus and its effect was unchanged in the presence of tetrodotoxin, atropine and indomethacin. The potency of chicken NMU-9 (EC50 360 +/- 60 nM; mean +/- S.E., n = 6) was approximately 8-fold less than that of pig NMU-8 (EC50 46 +/- 8 nM) but the maximum contraction produced by both agonists was not significantly different.

Evidence that neuromedin U may regulate gut motility in reptiles but not in mammals

Neuromedin U-8 induced a monophasic and concentration-dependent contraction of intact small intestine from the turtle, Pseudemys scripta, whereas the peptide had no effect upon the motility of rat and guinea pig gut. The maximum response produced by neuromedin U-8 was 56% of that produced by acetylcholine and 62% of that produced by potassium chloride. The potency and maximum response to neuromedin U-8 were unaffected by tetrodotoxin and atropine. The data suggest that neuromedin U may play a role in regulation of gut motility in lower vertebrates but not in mammals.

Vasopressin response in collecting ducts of rats resistant to mineralocorticoid hypertension.

In previous studies we found that vasopressin stimulation of both cyclic AMP (cAMP) formation in cortical collecting tubules (CCT) and sodium reabsorption in isolated perfused kidneys was markedly exaggerated in rats with mineralocorticoid hypertension. In the present study, we tested the response (cAMP accumulation) of cortical and outer medullary collecting tubules (OMCT) to vasopressin in two rat models that are resistant to deoxycorticosterone acetate (DOCA)-induced hypertension, the Wistar-Furth strain and NaCl-deficient rats. The blood pressure of normal outbred Wistar rats rose to hypertensive levels (systolic pressure more than 165 mm Hg) during a 5-week treatment with DOCA (10 mg/week) and 1% saline to drink. Significant hypertrophy of the heart and kidneys was also observed. Vasopressin (10(-8) M)-induced cAMP formation was enhanced 3.4-fold in the CCT (OMCT unchanged) of hypertensive rats compared with normotensive controls. Significant hypertrophy (as indexed by tubule diameter) of the CCT but not the OMCT was also observed in DOCA-salt hypertensive rats. Restriction of dietary NaCl (0.13% in chow, tap water to drink) completely prevented DOCA-induced hypertension, organ and CCT hypertrophy, and enhancement of vasopressin-stimulated cAMP formation in the CCT. In Wistar-Furth rats, DOCA-salt treatment did not alter blood pressure or cause significant organ hypertrophy. However, DOCA-salt treatment enhanced vasopressin-stimulated cAMP formation by 4.1-fold in CCT of Wistar-Furth rats, with significant tubular hypertrophy in the CCT but not the OMCT. We conclude that DOCA-induced hypertension and changes in CCT function are dependent on excess dietary NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and pharmacological characteristics of the gerbil α1a-adrenergic receptor

The spiral modiolar artery supplies blood and essential nutrients to the cochlea. Our previous functional study indicates the α(1A)-adrenergic receptor subtype mediates vasoconstriction of the gerbil spiral modiolar artery. Although the gerbil cochlea is often used as a model in hearing research, the molecular and pharmacological characteristics of the cloned gerbil α(1a)-adrenergic receptor have not been determined. Thus we cloned, expressed and characterized the gerbil α(1a)-adrenergic receptor and then compared its molecular and pharmacological properties to those of other mammalian α(1a)-adrenergic receptors. The cDNA clone contained 1404 nucleotides, which encoded a 467 amino acid peptide with a deduced sequence having 96.8, 96.4 and 91.6% identity to rat, mouse and human α(1a)-receptors, respectively. We transiently transfected the α(1a)-adrenergic receptor into COS-1 cells and determined its pharmacological characteristics by [(3)H]prazosin binding. Unlabeled prazosin had a K(i) of 0.89±0.1nM. The α(1A)-adrenergic receptor-selective antagonists, 5-methylurapidil and WB-4101, bound with high affinity and had K(i) values of 4.9±1 and 1.0±0.1nM, respectively. BMY-7378, an α(1D)-adrenergic receptor-selective antagonist, bound with low affinity (260±60nM). The 91.6% amino acid sequence identity and K(i)s of the cloned gerbil α(1a)-adrenergic receptor are similar to those of the human α(1a)-adrenergic receptor clone. These results show that the gerbil α(1a)-adrenergic receptor is representative of the human α(1a)-adrenergic receptor, lending validity to the use of the gerbil spiral modiolar artery as a model in studies of vascular disorders of the cochlea.

Respiratory dysfunction progresses with age in Kcna1-null mice, a model of sudden unexpected death in epilepsy

Increased breathing rate, apnea, and respiratory failure are associated with sudden unexpected death in epilepsy (SUDEP). We recently demonstrated the progressive nature of epilepsy and mortality in Kcna1−/− mice, a model of temporal lobe epilepsy and SUDEP. Here we tested the hypothesis that respiratory dysfunction progresses with age in Kcna1−/− mice, thereby increasing risk of respiratory failure and sudden death (SD).

Effects of Environmental Enrichment on Nicotine Addiction

The aim of this chapter is to summarize the research investigating the effects of environmental enrichment on the behavioral and neural effects of nicotine and integrate the limited nicotine data with the larger existing data of how environmental enrichment alters sensitivity to other abused psychostimulants. Environmental enrichment decreases the sensitivity to both acute and repeated nicotine-induced locomotor stimulatory effects. Also, environmental enrichment decreases the sensitivity to the discriminative stimulus properties of nicotine similar to those seen with amphetamine. Likewise, environmental enrichment appears to increase sensitivity to nicotine conditioned place preference, similar to what has been found previously with amphetamine. The ability of environmental enrichment to alter the neural effects of nicotine appears to be mediated through changes in dopamine transporter function in the medial prefrontal cortex and nucleus accumbens. Additionally, data indicate that enrichment may also alter the nicotinic acetylcholine receptors in key mesolimbic structures involved in abuse.