Biomedical subjects

Kirk P Conrad

Publications and source records attributed to Kirk P Conrad.

22 records · Page 2Linked to original sources

PubMed2003

Essential role for vascular gelatinase activity in relaxin-induced renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small arteries.

During pregnancy, relaxin stimulates nitric oxide (NO)-dependent renal vasodilation, hyperfiltration and reduced myogenic reactivity of small renal arteries via the endothelial ETB receptor subtype. Our objective in this study was to elucidate the mechanisms by which relaxin stimulates the endothelial ETB receptor/NO vasodilatory pathway. Using chronically instrumented conscious rats, we demonstrated that a specific peptide inhibitor of the gelatinases MMP-2 and -9, cyclic CTTHWGFTLC (cyclic CTT), but not the control peptide, STTHWGFTLS (STT), completely reversed renal vasodilation and hyperfiltration in relaxin-treated rats. Comparable findings were observed with a structurally different and well-established, general antagonist of MMPs, GM6001. In contrast, phosphoramidon, an inhibitor of endothelin-converting enzyme, did not significantly change the renal vasodilatory response to relaxin administration. When small renal arteries were incubated with either of the general MMP inhibitors, GM6001 or TIMP-2 (tissue inhibitor of MMP), or with the specific gelatinase inhibitor, cyclic CTT, the reduced myogenic reactivity of these blood vessels from relaxin-treated nonpregnant and midterm pregnant rats was totally abolished. Moreover, a neutralizing antibody specific for MMP-2 completely abrogated the reduced myogenic reactivity of small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats. In contrast, phosphoramidon did not significantly affect the reduction in myogenic reactivity. Using gelatin zymography, we showed increased pro and active MMP-2 activity in small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats relative to the control animals. Thus, inhibitors of MMPs in general and of gelatinases in particular reverse the renal vascular changes induced by pregnancy or relaxin administration to nonpregnant rats. Finally, the typical reduction in myogenic reactivity of small renal arteries from relaxin-treated nonpregnant rats was absent in ETB receptor-deficient rats, despite an increase in vascular MMP-2 activity. These results indicate an essential role for vascular gelatinase, which is in series with, and upstream of, the endothelial ETB receptor/NO signaling pathway in the renal vasodilatory response to relaxin and pregnancy.

Animals↗

PubMed2003

Time course and dose response of relaxin-mediated renal vasodilation, hyperfiltration, and changes in plasma osmolality in conscious rats.

The pregnancy hormone relaxin elicits renal vasodilation, hyperfiltration, and osmoregulatory changes when chronically administered to conscious, nonpregnant rats. The objective in this study was to determine the dose response and time course of hormone action, as well as the time required for recovery on stopping its administration. The threshold dose of recombinant human relaxin (rhRLX) for renal vasodilation and reduction in plasma osmolality was 0.15 microg/h when given by subcutaneous osmotic minipump for 2 days (an infusion rate that achieved circulating levels of approximately 6 ng/ml). The peak response was observed during the 0.4 microg/h infusion rate (serum rhRLX of approximately 11 ng/ml), which was comparable to our previous work using a 4.0 microg/h (serum rhRLX of approximately 20 ng/ml). In contrast, a dose of 40 microg/h was ineffective (serum rhRLX of approximately 80 ng/ml). When 4.0 microg/h rhRLX was administered by osmotic minipump for shorter periods (</=24 h), renal circulatory and osmoregulatory changes were observed by </=6 h. After removal of the osmotic minipump, these changes persisted for at least 12 h, but they were fully restored by 24 h. Even briefer administration of 4.0 microg/h rhRLX by intravenous infusion showed an onset of action in the kidney by 1-2 h. In contrast, the 40 microg/h dose of rhRLX elicited minimal effects, and comparable to our earlier report, 4.0 microg/h purified porcine relaxin was also relatively ineffective during short-term intravenous administration. In conclusion, the effect of relaxin on the renal circulation and osmoregulation is biphasic, insofar as high doses are relatively inactive, and the onset of action is more rapid than previously believed. These findings may be important to consider when evaluating relaxin in the treatment of renal disease.

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PubMed2002

Citrulline is not the major product using the standard "NOS activity" assay on renal cortical homogenates.

A standard approach to assessing nitric oxide synthase (NOS) activity in tissue homogenates is 1) removal of small-molecular-weight substances by size-exclusion chromatography, 2) adding back of substrates/cofactors in precise concentrations with a radioactive isotope of arginine (Arg), and 3) quantification of labeled citrulline (Cit) after separation of Arg and Cit by cation-exchange column chromatography. Using this approach and L-[2,3-3H]Arg, we found that the major product(s) was not Cit in cortical homogenates prepared from rat, mouse, and human kidneys. The product(s) mimicked Cit, insofar as it passed freely through cation-exchange columns and comigrated with Cit on both one-dimensional and two-dimensional straight-phase thin-layer chromatography systems. However, it was clearly resolved from Cit by precolumn derivatization and reverse-phase HPLC. The maximum velocity and Michaelis-Menten constant were approximately 100 pmol x mg protein(-1) x min(-1) and 100 microM, respectively, in renal cortical homogenates from rats. The enzyme activity was the same in the presence or absence of cofactors including Ca2+, calmodulin, tetrahydrobiopterin, and NADPH. It was only modestly inhibited by L-Arg analogs and was mainly in the supernatant after a 100,000 g centrifugation. These enzyme characteristics contrasted markedly with those simultaneously obtained for NOS activity in placental homogenates. Thus results from the conventional NOS activity assay should be viewed cautiously.

Animals↗

PubMed2002

Myogenic reactivity is reduced in small renal arteries isolated from relaxin-treated rats.

Administration of the ovarian hormone relaxin to nonpregnant rats vasodilates the renal circulation comparable to pregnancy. This vasodilation is mediated by endothelin (ET), the ET(B) receptor, and nitric oxide. Furthermore, endogenous relaxin mediates the renal vasodilation and hyperfiltration that occur during gestation. The goal of this study was to investigate whether myogenic reactivity of small renal and mesenteric arteries is reduced in relaxin-treated rats comparable to the pregnant condition. Relaxin or vehicle was administered to virgin female Long-Evans rats for 5 days at 4 microg/h, thereby producing midgestational blood levels of the hormone. The myogenic responses of small renal arteries (200-300 microm in diameter) isolated from these animals were evaluated in an isobaric arteriograph system. Myogenic reactivity was significantly reduced in the small renal arteries from relaxin-treated compared with vehicle-treated rats. The reduced myogenic responses were mediated by the ET(B) receptor and nitric oxide since the selective ET(B) receptor antagonist RES-701-1 and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester restored myogenic reactivity to virgin levels. The influence of relaxin was not limited to the renal circulation because myogenic reactivity was also reduced in small mesenteric arteries isolated from relaxin-treated rats. Thus relaxin administration to nonpregnant rats mimics pregnancy, insofar as myogenic reactivity of small renal and mesenteric arteries is reduced in both conditions.

Animals↗