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D K Little

Publications and source records attributed to D K Little.

9 recordsLinked to original sources
PubMed1994

A rapid and versatile method for screening endothelin converting enzyme activity.

A flexible two-step protocol has been developed for high-throughput assays of endothelin converting enzyme (ECE) activity. The method is illustrated using endothelial cell monolayers and crude cell extracts as enzyme sources for conversion of exogenous substrates (big endothelin isopeptides) to their respective vasoactive products (endothelin isopeptides). Cleavage conditions have been optimized to minimize substrate utilization (reducing cost) while maintaining sensitivity and selectivity for mature endothelin detection in receptor- and antibody-based assays. Endothelins are quantified in a separate step and comparable estimates of mature endothelin formation are obtained using both EIA and radioreceptor assays in 96-well formats. Human umbilical vein (HUVEC) and human aorta (HAEC) endothelial cells characterized by this method are found to preferentially convert the big endothelin-1 (big ET-1) isopeptide through a membrane-bound, thiorphan-insensitive, and phosphoramidon-sensitive zinc metalloendopeptidase. Both intact cells and membrane preparations used as the enzyme source predict similar IC50 values for phosphoramidon inhibition of ECE (ca 1 microM). The procedure described is simple, rapid, and suitable for high-volume screening.

Animals↗
PubMed1992

The effects of novel cathepsin E inhibitors on the big endothelin pressor response in conscious rats.

The aspartic protease, cathepsin E, has been shown to specifically cleave big endothelin (big ET-1) at the Trp21-Val22 bond to produce endothelin (ET-1) and the corresponding C-terminal fragment. To determine whether cathepsin E is a physiologically relevant endothelin converting enzyme (ECE), three novel and potent inhibitors of cathepsin E were administered to conscious rats prior to a pressor challenge with big ET-1. One of the inhibitors of cathepsin E, SQ 32,056 (3 mg/kg i.v.), blocked the big ET-1 response. However, this dose of SQ 32,056 also blocked the pressor response to ET-1. Phosphoramidon specifically inhibited the Big ET-1 pressor response. These results suggest that ECE is not cathepsin E.

Amino Acid Sequence↗
PubMed1991

Antihypertensive and renal activity of SQ 28,603, an inhibitor of neutral endopeptidase.

SQ 28,603 is a potent and selective inhibitor of neutral endopeptidase 3.4.24.11 (NEP), an enzyme that degrades atrial natriuretic peptide (ANP). In conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rats, 300 mumol/kg, i.v., of SQ 28,603 significantly lowered mean arterial pressure (MAP) from 177 +/- 12 to 154 +/- 8 mm Hg and increased urinary cyclic guanosine monophosphate (GMP) excretion from 204 +/- 70 to 1,068 +/- 326 pmol/kg/min within 2 h. Urinary sodium excretion increased within 20 min from a control 51.2 +/- 17.3 to 102.1 +/- 26.7 muEq/kg/min. Infusion of SQ 28,603 (3.7 mumol/kg/min) for 6 h in a separate group of conscious DOCA/salt hypertensive rats gradually reduced MAP from 180 +/- 7 to 142 +/- 7 mm Hg and increased urinary cyclic GMP excretion from 182 +/- 36 pmol/kg/min to 1,009 +/- 394 pmol/kg/min. Despite the continuous infusion of the inhibitor, the natriuretic response peaked during the first hour of treatment at 128 +/- 18 muEq/kg/min (vehicle = 54 +/- 10 muEq/min). Plasma ANP was significantly greater in the rats infused with SQ 28,603 than in those receiving vehicle (333 +/- 108 and 98 +/- 14 fmol/ml, respectively). SQ 28,603 also significantly reduced NEP activity by 95% in the kidneys (1.28 +/- 0.08 vs. 18.35 +/- 0.61 mumol/min after SQ 28,603 and vehicle respectively) and by 77% in the lungs (0.29 +/- 0.03 vs. 0.92 +/- 0.14 mumol/kg after SQ 28,603 and vehicle, respectively). In conclusion, inhibition of NEP activity by SQ 28,603 significantly decreased MAP and increased plasma ANP concentrations and urinary excretion of cyclic GMP in conscious DOCA/salt hypertensive rats.

Alanine↗
PubMed1991

Possible regulation of atrial natriuretic factor by neutral endopeptidase 24.11 and clearance receptors.

The effects of a clearance receptor ligand Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys-NH2 with a disulfide bridge between the two cycteines [C-ANF(4-23)] and the potent neutral endopeptidase (NEP) inhibitor SQ 28,603 on mean arterial pressure (MAP), plasma atrial natriuretic factor (ANF) concentration and renal excretion of sodium and cyclic GMP were determined in conscious deoxycorticosterone acetate/salt hypertensive rats and normotensive rats. In the hypertensive rats, i.v. infusion of C-ANF(4-23) produced depressor responses of approximately 25 mm Hg, but did not significantly affect plasma ANF concentration or stimulate cyclic GMP excretion. In contrast, SQ 28,603 (300 mumol/kg i.v.) significantly reduced MAP and increased excretion of sodium and cyclic GMP. When C-ANF(4-23) was administered in combination with SQ 28,603, the depressor activity was additive and plasma ANF concentrations were significantly increased. The excretion of cyclic GMP was slightly enhanced, but, was not significantly different from the effects of SQ 28,603 alone. Neither SQ 28,603 nor C-ANF(4-23) affected MAP or plasma ANF in the normotensive rats. Finally, the in vitro hydrolysis of C-ANF(4-23) by NEP was prevented by SQ 28,603, indicating that inhibition of NEP may protect peptides recognized by the clearance receptors as well as the biological receptors for ANF. Therefore, the additive effects of C-ANF(4-23) and SQ 28,603 may be due to blockade of separate pathways which inactivate ANF or to the inhibition of C-ANF(4-23) degradation by NEP.

Alanine↗
PubMed1990

Renal and depressor effects of SQ 29,072, a neutral endopeptidase inhibitor, in conscious hypertensive rats.

The depressor and renal responses to the neutral endopeptidase (NEP) inhibitor, SQ 29,072, were characterized in both the conscious spontaneously hypertensive rat (SHR) and the conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rat. Inhibition of tissue NEP activity by pharmacologically active doses was also ascertained in both hypertensive models. Intravenous administration of 300 mumol/kg of SQ 29,072 significantly reduced mean arterial pressure (MAP), produced modest natriuretic and diuretic responses, and inhibited renal NEP activity by approximately 40% in conscious SHR. Doses of 100 and 300 mumol/kg of SQ 29,072 elicited greater depressor responses (-36 +/- 7 and -41 +/- 8 mm Hg, respectively) in DOCA/salt hypertensive rats than in SHR (-11 +/- 24 and -31 +/- 5 mm Hg, respectively). SQ 29,072 (300 mumol/kg, i.v.) also inhibited renal NEP activity to a greater extent (70%) in DOCA/salt hypertensive rats. Similarly, the depressor responses to exogenous ANP 99-126 (1, 3, and 10 nmol/kg, i.v.) were greater in DOCA/salt hypertensive rats (-16 +/- 4, -38 +/- 6, and -73 +/- 6 mm Hg, respectively) than in the SHR (0 +/- 6, -17 +/- 3, and -24 +/- 3 mm Hg, respectively). Finally, equidepressor doses of SQ 29,072 and ANP 99-126 both increased urine volume as well as sodium and cyclic GMP excretion in conscious DOCA/salt hypertensive rats. In conclusion, the profile of depressor and renal activities produced by SQ 29,072 was consistent with potentiation of endogenous ANP by inhibition of NEP in conscious SHR and DOCA/salt hypertensive rats.

Amino Acids↗
PubMed1989

Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide.

Acetylation↗
PubMed1987

Calmodulin antagonists inhibit latch bridges in detergent skinned swine carotid media.

Ca2+-calmodulin-dependent phosphorylation of the 20,000 apparent molecular weight (Mr) myosin light chain (MLC) is widely believed to be the primary regulator of smooth muscle stress development. Stress maintenance, however, has been hypothesized to be the result of a Ca2+-dependent state called latch, defined as stress maintenance by slowly cycling, dephosphorylated latch bridges. This study was designed to examine the role of calmodulin in the Ca2+ dependence of latch. Swine carotid medial fibers were detergent skinned with Triton X-100 and MLC phosphorylation levels were measured by two-dimensional gel electrophoresis. Experiments were performed in which calmodulin antagonists, W-7 or trifluoperazine (TFP), were used to prevent Ca2+-dependent contractions or to relax previously contracted muscle strips. Both protocols resulted in similar decreases in stress with MLC phosphorylation levels correlating to the level of stress under all conditions. In another series of experiments, the skinned fibers were first contracted with Ca2+ and ATP and then exposed to Ca2+ and cytidine 5'-triphosphate (CTP). This resulted in stress maintenance with basal levels of MLC phosphorylation. W-7 relaxed Ca2+ and CTP maintained stress in a concentration-dependent manner. The potency of W-7 for relaxation of the CTP maintained stress was similar to that for inhibition of the ATP developed stress determined in the first set of experiments. The results demonstrate that calmodulin antagonists inhibit stress associated with MLC phosphorylation and also stress independent of MLC phosphorylation. This suggests that a calmodulin-like, Ca2+-binding protein may be important for latch bridge attachment.

Adenosine Triphosphate↗