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R M Edwards

Publications and source records attributed to R M Edwards.

At least 91 records · Page 5Linked to original sources
PubMed1990

Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance.

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.

Binding, Competitive↗
PubMed1990

Calcitonin gene-related peptide: effects on renal arteriolar tone and tubular cAMP levels.

The effects of calcitonin gene-related peptide (CGRP) and salmon calcitonin (SCT) on isolated afferent and efferent arteriolar tone and on adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in isolated tubules from rabbit kidney were compared. By themselves, CGRP and SCT had no effect on arteriole diameter. In norepinephrine-contracted afferent arterioles, CGRP, but not SCT, produced a concentration-dependent relaxation [50% maximal dose, (EC50) = 1.3 nM]. In contrast, neither CGRP nor SCT had any effect on efferent arterioles precontracted with norepinephrine or angiotensin II. CGRP, but not SCT, stimulated cAMP accumulation in glomeruli (EC50 = 0.5 nM). In tubules, CGRP increased cAMP levels only in SCT-responsive segments, e.g., cortical and medullary thick ascending limbs and distal convoluted tubules. In the medullary thick ascending limb, CGRP was approximately 500-fold less potent than SCT in stimulating cAMP accumulation with EC50s of 0.22 microM and 0.41 nM, respectively. The effect of maximum concentrations (1 microM) of CGRP and SCT on cAMP levels in the medullary thick ascending limb were not additive. The results suggest that there are specific CGRP receptors on afferent arterioles that produce relaxation and in glomeruli that are associated with an increase in cAMP production. In tubules, CGRP appears to be a weak agonist at the SCT receptor. We conclude that CGRP may play a role in the regulation of renal hemodynamics.

Animals↗
PubMed1990

Renal microvascular effects of endothelin.

The effects of endothelin 1, 2, and 3 (ET-1, -2, -3) on lumen diameter of individual afferent and efferent arterioles dissected from rabbit kidney were examined. ET-1 produced concentration-dependent and long-lasting decreases in lumen diameter in both arterioles. The 50% maximum response (EC50) values were 1.4 +/- 0.41 and 0.9 +/- 0.65 nM for afferent and efferent arterioles, respectively. In afferent arterioles, ET-2 produced decreases in lumen diameter (EC50 = 3.3 +/- 1.75 nM) that were indistinguishable from ET-1. However, ET-3 was considerably less potent (EC50 = 21.9 +/- 6.0 nM, P less than 0.05) than ET-1 or ET-2. Similar results were obtained in the efferent arteriole in which the EC50 for ET-2 (0.25 +/- 0.1 nM) was similar to ET-1, but ET-3 was significantly less potent (EC50 = 2.6 +/- 0.4 nM, P less than 0.05). Nicardipine (0.01-1 microM) produced concentration-dependent shifts in the ET-1 concentration-response curve in afferent arterioles. Verapamil (1 microM) also caused a significant shift in the ET-1 response curve. The contractile response to ET-1 was significantly more sensitive to nicardipine than was the response to norepinephrine. In contrast, the response of efferent arterioles to ET-1 and norepinephrine was unaffected by nicardipine or verapamil. The results demonstrate that ETs are potent vasoconstrictors of both the pre- and postglomerular microvasculature and may play a role in the regulation of renal hemodynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effect of enalapril or the thromboxane receptor antagonist, daltroban, in rats with subtotal renal ablation.

There is evidence to suggest that thromboxane synthesis inhibition will attenuate the hypertension and proteinuria associated with subtotal renal ablation. In the present study, the thromboxane receptor antagonist, daltroban, (30 mg/kg/day i.p.) or vehicle was administered to rats for 3 weeks starting 2 weeks after partial renal ablation (right uninephrectomy and ligation of approximately two-thirds of the blood supply to the left kidney). Renal ablation was associated with proteinuria and increased systolic blood pressure. Neither the proteinuria nor the hypertension was affected by daltroban administration. Histological examination of the remaining kidney demonstrated no beneficial effect of daltroban. In a second study, it was determined that, 2 weeks after renal ablation, urinary thromboxane excretion was significantly increased, and subsequent administration of daltroban for 2 weeks resulted in significant blockade of the effects of the thromboxane mimetic, U46619. In a third study, enalapril (50 mg/l in the drinking water) resulted in a significant attenuation of the proteinuria, hypertension and glomerular lesions associated with partial renal ablation. The data indicate that enalapril, but not daltroban, protects against the development of renal disease associated with reduced renal mass.

Animals↗
PubMed1989

Renal microvascular effects of vasopressin and vasopressin antagonists.

The effects of vasopressin (AVP) and vasopressin antagonists on lumen diameters of cortical afferent and efferent arterioles isolated from rabbit kidneys were examined. Over a concentration range of 10(-14) to 10(-7) M, AVP had no effect on lumen diameters of afferent arterioles, although the arterioles were responsive to norepinephrine. Similarly, addition of 10(-8) M AVP to the lumen of afferent arterioles or to the bath of arterioles pretreated with indomethacin had no effect. In contrast, AVP caused a concentration-dependent reduction of lumen diameters of efferent arterioles. AVP was approximately 100-fold more potent than norepinephrine in producing contraction of efferent arterioles. The V1-selective antagonist, [d(CH2)5Tyr(Me)]AVP, and the V1/V2-antagonist, d(CH2)5D-Tyr(Et) desGlyVAVP, inhibited the vasoconstriction produced by AVP in a concentration-dependent but noncompetitive manner. The V2-selective antagonist, [d(CH2)5D-Ile]VAVP, had no significant effect on AVP-induced vasoconstriction. We conclude that, under the in vitro conditions used, AVP selectively contracts efferent arterioles. The results provide direct evidence for a postglomerular vascular effect of AVP in the renal cortex. This activity, together with its previously described effects on the glomerulus, suggests that AVP may produce changes in glomerular function and/or peritubular forces that are involved in tubular reabsorption.

Animals↗
PubMed1988

The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway.

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.

Alcohol Oxidoreductases↗
PubMed1988

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the nephron.

The effect of vasoactive intestinal peptide (VIP) on adenylate cyclase activity in individual segments of the rabbit nephron was examined. VIP caused a significant increase in adenylate cyclase activity in the distal convoluted tubule and in the cortical and medullary portions of the collecting tubule. VIP also caused a concentration-dependent increase in adenylate cyclase activity in rat and dog collecting tubules. The results demonstrate that VIP has a direct stimulatory effect on tubular adenylate cyclase which may be involved in the increase in electrolyte excretion observed with VIP.

Adenylyl Cyclases↗
PubMed1988

Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair.

The synthesis of a gene for the HIV TAT protein is described using a novel approach that capitalises on the ability to synthesise oligonucleotides of greater than 100 bp in length. It involves the synthesis of large oligomers covering one strand of the desired gene in its entirety and the use of small complementary bridging and adapter oligonucleotides to direct the assembly and cloning of the large oligomers. After ligation to the cloning vector the partially single stranded intermediate is transformed directly into the recipient bacterial host where the plasmid is repaired. The synthetic tat gene has been expressed in HeLa cells and is shown to trans-activate TAR+ but not TAR- HIV LTR-CAT constructs.

Amino Acid Sequence↗
PubMed1988

Characterization of alpha-adrenoceptors on isolated rabbit renal arterioles.

Postsynaptic alpha-adrenoceptors were characterized in afferent and efferent arterioles isolated from rabbit renal cortex. In both the afferent and efferent arteriole the selective alpha 1-adrenoceptor agonists phenylephrine and cirazoline produced concentration-dependent vasoconstrictor responses with the maximum responses being equal to that of norepinephrine. The selective alpha 2-receptor agonists B-HT 933 and UK 14304 were examined over a concentration range of 10(-9) to 10(-4) M. At these concentrations, they were either inactive (B-HT 933) or produced maximum contractile responses only 60% (UK 14304) of that produced by norepinephrine or the alpha 1-receptor agonists. Prazosin (10(-7) M), an alpha 1-receptor antagonist, produced a rightward shift in the concentration-response curve to norepinephrine, yielding apparent dissociation constants of 1.7 and 1.2 X 10(-8) M for the afferent and efferent arteriole, respectively. Prazosin also antagonized the contractile effects of UK 14304 (apparent dissociation constants, 0.9 and 1.1 X 10(-8) M for afferent and efferent arterioles, respectively). The selective alpha 2-receptor antagonist, rauwolscine (10(-7) M), had no effect on norepinephrine-mediated vasoconstriction. These results confirm the presence of alpha-adrenoceptors on the glomerular arterioles that mediated vasoconstriction. Furthermore, these receptors appear to be exclusively of the alpha 1-subtype.

Animals↗
PubMed1988

Mechanism of alpha 2-adrenoceptor agonist-induced diuresis.

In vivo and in vitro studies were performed to assess the mechanism of the diuretic effect of B-HT 933, a selective alpha 2-adrenoceptor agonist. In conscious Sprague-Dawley rats whose plasma vasopressin (AVP) levels were increased by infusion of hypertonic NaCl, B-HT 933 had no effect on AVP secretion. In Brattleboro homozygous (DI) rats, the antidiuretic dose response to AVP was shifted to the right by B-HT 933. In addition, a sustained antidiuresis induced in rats by infusion of 10 pg/min AVP was attenuated by B-HT 933 in a concentration-dependent manner. Pretreatment of DI rats with pertussis toxin (2 micrograms/kg iv) 4-5 days before testing abolished the inhibitory effect of B-HT 933 on AVP-induced antidiuresis. In outer medullary collecting ducts of DI rats, norepinephrine and B-HT 933 produced significant inhibition of AVP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. In contrast, the selective alpha 1-adrenoceptor agonist cirazoline had no effect on AVP-induced cAMP formation. The inhibitory effect of norepinephrine was antagonized by the selective alpha 2-adrenoceptor antagonist rauwolscine but not by prazosin, a selective alpha 1-antagonist. In outer medullary collecting ducts dissected from the pertussis toxin-treated DI rats used in the in vivo studies, the inhibitory effect of norepinephrine and B-HT 933 on AVP-stimulated cAMP accumulation was abolished. The results indicate that the hydrosmotic action of AVP is inhibited by alpha 2-agonists via a pertussis toxin-sensitive mechanism.

Adrenergic alpha-Agonists↗
PubMed1988

Inhibition of vasopressin-stimulated cyclic AMP accumulation by alpha-2 adrenoceptor agonists in isolated papillary collecting ducts.

The effects of selective alpha adrenoceptor agonists and antagonists on vasopressin (VP)-sensitive cyclic AMP (cAMP) formation in microdissected rat papillary collecting ducts were examined. In the presence of 10(-10) M VP, norepinephrine and the selective alpha-2 adrenoceptor agonist, B-HT 933, produced almost total inhibition of VP-stimulated cAMP accumulation. Half-maximal inhibition occurred at 1 x 10(-8) M and 6 x 10(-7) M for norepinephrine and B-HT 933, respectively. Cirazoline, a selective alpha-1 adrenoceptor agonist, had no significant effect on VP-stimulated cAMP accumulation. The inhibitory effects of norepinephrine and B-HT 933 were antagonized by rauwolscine but not by prazosin. The antagonism of B-HT 933-induced inhibition of VP-stimulated cAMP accumulation was competitive with an antagonist dissociation constant (KB) of 10.9 x 10(-9) M. Preincubation of papillary collecting ducts with pertussis toxin (1 microgram/ml for 1 hr at 37 degrees C) attenuated, by 65%, the inhibitory effect of B-HT 933 on VP-stimulated cAMP levels. These results demonstrate that alpha-2 adrenoceptors capable of inhibiting VP action are present on the papillary collecting duct. Furthermore, the alpha-2 adrenoceptor-induced inhibition of VP-stimulated cAMP accumulation is pertussis-toxin sensitive. This suggests that alpha-2 adrenoceptors are coupled negatively to adenylate cyclase, via the guanine nucleotide binding protein, in the collecting tubule.

Adenylate Cyclase Toxin↗
PubMed1987

The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.

The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes.

Alcohol Oxidoreductases↗
PubMed1987

Fluid compartmentation in skeletal muscle and carcass of Mus musculus acclimated to water scarcity.

1. Total water (TW), and extracellular water (ECW) (as sodium and chloride space) were determined in skeletal muscle and carcass of Mus musculus acclimated to long-term water shortage. 2. The presence of fat in control mice and those in early stages of acclimation resulted in an apparent increase in TW and ECW as acclimation proceeded. 3. In contrast, fluid volumes per fat-free weight were either unchanged from controls or reduced. 4. Sodium space exceeded chloride space. 5. Muscle and carcass had essentially the same pattern of fluid shifts. 6. We conclude that ECW maintenance is a preeminent component of the acclimation process in this species.

Adaptation, Physiological↗
PubMed1987

Lack of effect of atriopeptin II on rabbit glomerular arterioles in vitro.

This study was designed to determine if atriopeptin II (AP II) has any direct action on glomerular arterioles in vitro. Single superficial afferent or efferent arterioles were dissected from rabbit kidney, cannulated with micropipettes, and changes in lumen diameter were measured. The effect of AP II (10-(12)-10(-7) M) was tested alone or in arterioles precontracted with norepinephrine (3 X 10(-7) M, afferent) or angiotensin II (10(-10) M, efferent). By itself, AP II had no effect on lumen diameter in afferent or efferent arterioles, but both arterioles responded to 3 X 10(-7) M norepinephrine with 74 and 54% reductions in lumen diameter, respectively. Similarly, in norepinephrine-contracted afferent arterioles and angiotensin II-contracted efferent arterioles, AP II did not alter lumen diameter although acetylcholine (10(-6) M) produced near maximal relaxation of both arterioles. Furthermore, luminal addition of AP II (10(-7) M) had no effect on control lumen diameter or on the response to norepinephrine or angiotensin II. The preparation of AP II was biologically active since it caused dose-dependent increases in guanosine 3',5'-cyclic monophosphate levels in isolated rat glomeruli. These results suggest that the renal vascular effects of AP II observed in vivo are not due to a direct action on superficial glomerular arterioles but rather to an, as yet, unidentified indirect action of this peptide.

Angiotensin II↗
PubMed1987

Thyrotrophin releasing hormone--5-hydroxytryptamine interactions in the brain studied using chronic immunization and chemical lesioning techniques.

This study investigates the effects of chemically lesioning 5-hydroxytryptamine (5HT) neurones and chronic passive immunization of central thyrotrophin releasing hormone (TRH) on 5HT and TRH mediated behavioural responses. 5HT lesions produced by 5,7-dihydroxytryptamine (5,7-DHT) enhanced the behavioural response produced by the 5HT receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MEODMT) while decreasing the locomotor hyperactivity observed following administration of the TRH analogue CG 3509 but having no effect on the reversal of pentobarbitone sleep-time produced by CG 3509. Chronic intracerebroventricular infusion of the purified TRH antibody markedly increased the length of pentobarbitone-induced sleep-time while enhancing the effects of CG 3509 both on locomotor activity and pentobarbitone-induced sleep. TRH antibody infusion also increased the response produced by 5-MEODMT. The results indicate that chronic passive immunization of central TRH induces changes in TRH receptor responsiveness and that there is a functional interaction between TRH and 5HT neuronal systems.

Animals↗
PubMed1986

Comparison of the effects of fenoldopam, SK & F R-87516 and dopamine on renal arterioles in vitro.

The ability of the selective DA1 agonists fenoldopam and SK & F R-87516 to relax norepinephrine-contracted afferent and efferent arterioles isolated from rabbit kidney was compared. In both arterioles, fenoldopam and SK & F R-87516 produced a concentration-dependent relaxation with a potency similar to that of dopamine. Fenoldopam also relaxed efferent arterioles contracted with angiotensin II. The selective DA1 receptor antagonist, SK & F R-83566, caused parallel displacements in the concentration-response curves to SK & F 82526, corresponding to dissociation constants of 10.5 and 8.3 nM for afferent and efferent arterioles, respectively. The results provide direct evidence that the dopamine receptor present on renal resistance vessels is of the DA1 subtype.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1986

The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.

Animals↗