CYP4T1--a cytochrome P450 expressed in rainbow trout (Oncorhynchus mykiss) liver.

Total RNA isolated from a rainbow trout (Oncorhynchus mykiss) liver was subjected to RT/PCR using degenerate primers designed from homologous regions amongst cytochrome P450 CYP4 proteins. PCR amplification resulted in a single electrophoretic band which was excised, purified and sequenced directly, using cycle sequencing. The deduced protein sequence demonstrated the closest amino acid identity to rabbit CYP4B1 (54.6%) and rat CYP4B2 (55.4%). Phylogenic analysis of this sequence was found to be significantly different to any other CYP4 sequence and has been named CYP4T1. This represents the first CYP4 family member to be identified in an aquatic vertebrate.

Isolation and identification of a cytochrome P450 sequence in an Australian termite, Mastotermes darwiniensis.

A partial cytochrome P450 sequence was RT/PCR amplified from total RNA isolated from the whole body of worker class termites (Mastotermes darwiniensis). The degenerate primers used were designed from conserved regions from 4 different species: rat, human, cockroach and drosophila. The sequence was defined by the presence of the typical P450 heme-binding region and invariant residues found in all P450 proteins. The deduced amino acid sequence is 67% identical to cockroach (Blaberus discoidalis) CYP4C1, with only 39% and 42% identity to either CYP4A1 or CYP4B1, respectively, and has been named CYP4C8. Similar low sequence homology was observed between the termite sequence and the mouse CYP3a16 (39%) and black-swallow butterfly CYP6B3 (41%) P450 proteins. The CYP4C8 sequence contains variations in the 13-residue sequence characteristic of family 4 members, distinct from those seen for CYP4D1 and CYP4F family members. M. darwiniensis has been proposed as the "missing link between cockroaches and termites," with the genus Mastotermes dating back some 20-40 million years. The phylogenetic distance between B. discoidalis and M. darwiniensis would suggest that CYP4C8 represents the most ancient of the CYP4 family members.

Chronic (-)-isoprenaline infusion down-regulates beta 1- and beta 2-adrenoceptors but does not transregulate muscarinic cholinoceptors in rat heart.

Regulation of beta-adrenoceptor (beta-ar) subtypes and transregulation of muscarinic cholinoceptors (mAchr) was examined in regions of rat heart after chronic infusion of (-)-isoprenaline (450 micrograms/kg per hour) for 14 days. Following (-)-isoprenaline infusion systolic blood pressure was reduced for 10 days but then gradually returned to control levels, whereas heart rate was increased for 7 days before declining to a level significantly above control. Heart weight to body weight ratio was increased in (-)-isoprenaline treated rats. beta-ar subtype densities were measured by quantitative autoradiography with [125I]-cyanopindolol (CYP) in sinoatrial node (SA), atrioventricular node (AV), bundle of His (BH), left (LB) and right (RB) bundle branches, interventricular (IVS) and interatrial (IAS) septa, right atria (RA), apex (AX) and mitral valve (MV). beta 1-ars were reduced by 59.1-74.2% in the AV conducting regions, 53.4% in the SA node and 43.3-53.4% in myocardial areas, beta 2-ars were markedly reduced in myocardial regions (93.2-98.5%) and in pacemaker and conducting regions (87.7-97.8%). No changes in mAchr densities measured using [3H]-N-methyl scopolamine (NMS) occurred in the AV node, BH, LB, RB, IVS and IAS following (-)-isoprenaline infusion. Densities of beta 1- and beta 2-ars and mAchrs were also measured in ventricular homogenates from control and (-)-isoprenaline treated animals. beta-ar levels were significantly reduced (P < 0.05) in treated animals and the ratio of beta 1- to beta 2-ars increased after treatment. mAchr density in ventricular homogenates measured using either [3H]-NMS or [3H]-quinuclidinyl [phenyl-4-3H]benzilate (QNB) was unchanged. Homogenates of left and right ventricle also showed no change using [3H]-NMS. Organ bath studies were used to investigate the effect of (-)-isoprenaline infusion on negative inotropic and chronotropic effects of the non-selective muscarinic receptor agonist bethanechol in left and right atria, respectively. Lower concentrations of bethanechol (3 x 10(-10) to 10(-6) M) produced a negative inotropic response in isolated electrically driven left atria from (-)-isoprenaline treated rats, but not from control rats, with the slope of the curves being significantly different between groups (ANCOVA, P = 0.037). At concentrations of bethanechol from 10(-6) to 3 x 10(-4) M the negative inotropic response was not changed between (-)-isoprenaline treated and control animals. Bethanechol also produced a negative chronotropic response at lower concentrations (10(-10) to 10(-6) M) in (-)-isoprenaline treated rats, but not in controls. A second, steeper phase of the negative chronotropic response occurred at concentrations of bethanechol greater than 10(-6) M and was also seen in control rats. Expression of M2 (cardiac) mAchrs (m2Achr) in left and right ventricular tissues measured using a quantitative non-competitive polymerase chain reaction (PCR) assay showed a significant (P = 0.001) 28.5% increase in expression in left ventricle and a significant (P = 0.003) 21.5% decrease in expression in right ventricle after (-)-isoprenaline treatment, compared to controls. There was no significant difference in total ventricular m2Achr expression between the two groups of rats. The results suggest that chronic beta-ar stimulation down-regulates both beta 1- and beta 2-ars, and appears to differentially transregulate m2Achr expression, but not mAchr protein. Following (-)-isoprenaline infusion, muscarinic receptor mediated responses were sensitised, with no change in receptor densities, suggesting changes occur in the cell signalling system beyond the level of the receptor.

Resistance vessel gene expression of nerve growth factor is elevated in young spontaneously hypertensive rats.

OBJECTIVE: In this study we determined whether the enhanced production of nerve growth factor (NGF) and the associated hypernoradrenergic innervation of the vasculature of the spontaneously hypertensive rat (SHR) was associated with an increased gene expression of messenger (m)RNA encoding for nerve growth factor. DESIGN: It has been shown previously that the hypernoradrenergic innervation of the SHR occurs early, as does the enhanced expression for NGF. In this study we analysed the content of NGF mRNA in blood vessels from young SHR and normotensive Wistar-Kyoto (WKY) rats. METHODS: Total RNA was isolated from mesenteric arteries from 2-, 10- and 43-day-old SHR and WKY rats and RNA was also isolated from caudal arteries from 43-day-old rats. The RNA was subjected to Northern transfer or slot blots and the content of NGF mRNA measured after hybridization with a 32P-labelled complementary (c)DNA probe for NGF. RESULTS: Slot blot analysis indicated a larger concentration of NGF mRNA in mesenteric and caudal arteries from SHR than for tissues from WKY rats. CONCLUSIONS: In this genetic model of hypertension the results indicate an association between an enhanced level of NGF mRNA and the appearance of vascular hypernoradrenergic hypertension.

Nerve growth factor mRNA content parallels altered sympathetic innervation in the spontaneously hypertensive rat.

1. In order to explore the mechanisms responsible for the hypernoradrenergic innervation of the vasculature in the spontaneously hypertensive rat (SHR) the tissue content of nerve growth factor messenger ribonucleic acid (NGFmRNA) was examined. 2. The concentration of NGFmRNA was markedly elevated in mesenteric veins obtained from SHR when compared with the contents of NGFmRNA in veins from Wistar Kyoto rats (WKY). 3. The NGFmRNA content of kidneys was greater in SHR when compared with the levels present in WKY rats for 10- and 43-day-old animals. 4. In contrast to the pattern observed for veins and kidneys, the NGFmRNA content of SHR hearts was smaller than those present in hearts from WKY rats for 2, 10 and 43-day-old animals. 5. The results demonstrate that tissues with enhanced innervation (the kidney and mesenteric vasculature) in SHR are associated with an enhanced expression of NGFmRNA. In contrast, the heart, which does not display an enhanced sympathetic innervation in the SHR, does not have an increased expression of NGFmRNA. 6. It is suggested that in the SHR there is a tight relationship between hypernoradrenergic innervation in the vasculature and gene expression for NGFmRNA.

Failure of propranolol to inhibit the epinephrine-induced enhancement of responses to sympathetic nerve stimulation in the rat mesenteric vascular bed.

Experiments were designed to characterize the nature of the epinephrine-induced potentiation of responses to sympathetic nerve stimulation in the Hooded Wistar rat. The responses to sympathetic nerve stimulation were determined in the isolated perfused mesenteric vascular bed preparation before and after infusion of epinephrine (at 0.27 or 2.7 microM); at the conclusion of the experiment the content of epinephrine in the mesenteric artery was determined. The intraluminal infusion of epinephrine at both high and low concentrations potentiated the responses of the preparation to sympathetic nerve stimulation. Mesenteric artery concentrations of this catecholamine were unchanged at the lower concentration (0.27 microM), but were increased after perfusion of epinephrine at the higher concentration (2.7 microM). The beta adrenoceptor antagonist propranolol (0.5 microM) did not prevent the epinephrine-associated potentiation of responses to sympathetic nerve stimulation, nor did it influence the pressor effects of exogenous norepinephrine. The results suggest that beta adrenoceptors do not play a role in the epinephrine-induced potentiation of responses to sympathetic nerve stimulation in the rat mesenteric vascular bed preparation. This potentiation may, however, be related to a desensitization of presynaptic inhibitory alpha adrenoceptors.