Relaxin mediates uterine artery compliance during pregnancy and increases uterine blood flow.

Normal pregnancy involves dramatic remodeling of the uterine vasculature, with abnormal vascular adaptations contributing to pregnancy diseases such as preeclampsia. The peptide hormone relaxin is important for the renal and systemic hemodynamic adaptations to pregnancy, and has been shown to increase arterial compliance and outward hypertrophic remodeling. Therefore, we investigated the possibility that relaxin acts on its receptor, RXFP1, to mediate uterine artery compliance in late pregnancy and increase uterine blood flow velocity in rats. RXFP1 was predominantly localized to the tunica media vascular smooth muscle cells in the uterine artery, although receptors were also detected in endothelial cells. Highest expression of Rxfp1 in the uterine artery occurred in estrus and early pregnancy. Isolated uterine arteries from late pregnant rats treated with a monoclonal antibody against circulating relaxin (MCA1) had significantly increased vessel wall stiffness compared with controls, with no reduction in wall thickness. Chronic infusion of relaxin (4 µg/h, osmotic minipump) for 5 d in nonpregnant rats significantly increased uterine artery blood flow velocity. Overall, these data demonstrate a functional role for relaxin in mediating uterine artery compliance in pregnant rats, which may be necessary to maintain adequate uterine blood flow to the uterus and placenta.

Increased Myogenic Reactivity of Uterine Arteries from Pregnant Rats with Reduced Uterine Perfusion Pressure.

The etiology of preeclampsia remains unknown. However, a contributing factor to this hypertensive disease of pregnancy is a reduction in uterine perfusion pressure resulting in placental ischemia. Uterine arteries may be a major regulator of this process through changes in vascular reactivity and localized blood flow. The reduced uterine perfusion pressure (RUPP) pregnant rat is an established animal model of preeclampsia pathology. Pregnant Sprague Dawley rats were used for this investigation and subjected to RUPP or SHAM surgery on Day 14 of gestation. On Day 21 of gestation, animals were terminated and resistance-caliber uterine arteries were harvested and mounted on a pressurized arteriograph to examine myogenic reactivity, agonist induced vasodilation (methacholine and VEGF), and vasoconstriction (phenylephrine and U-46619). Resistance-caliber uterine arteries from RUPP animals exhibited increased myogenic reactivity and decreased vasodilation (methacholine and VEGF) compared to SHAM uterine arteries (p<0.05). Phenylephrine and U-46619 induced constriction was similar in uterine arteries between RUPP and SHAM rats. These results suggest that resistancecaliber uterine arteries from RUPP pregnant rats are altered to reflect a more constrictive phenotype which may play a role in the development of maternal hypertension demonstrated in these animals and thereby potentially in preeclampsia.

Alterations in vasomotor systems and mechanics of resistance-sized mesenteric arteries from SHR and WKY male rats following in vivo testosterone manipulation.

BACKGROUND: Testosterone (T) and the sympathetic nervous system each contribute to the pathology of hypertension. Altered blood vessel reactivity is also associated with the pathology of high blood pressure. The purpose of this study was to examine the effects of T manipulation in the regulation of resistance-sized blood vessel reactivity. METHODS: Adult spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) male rats at 8 weeks of age were used. The rats were divided into groups consisting of gonadally intact controls (CONT), castrate with sham implant (CAST) and castrate with T implant (CAST + T) (n = 6 to 12 per group). Following a short-term period of T treatment (approximately 4 weeks), plasma norepinephrine (NE) and plasma T were assessed by performing high-performance liquid chromatography and RIA, respectively. Resistance-sized mesenteric artery reactivity was assessed on a pressurized arteriograph for myogenic reactivity (MYO), phenylephrine (PE) responsiveness and passive structural mechanics. RESULTS: SHR and WKY males exhibited similar physiological trends in T manipulation, with castration significantly lowering plasma T and NE and T replacement significantly increasing plasma T and NE. T manipulation in general resulted in significant alterations in MYO of second-order mesenteric arteries, with T replacement decreasing MYO in SHR (P < 0.05) compared to CONT, T replacement increasing MYO, and CAST decreasing MYO in WKY rats (P < 0.001) compared to CONT rats. Additionally, PE-induced constriction was significantly altered in both strains following T treatment, with the effective concentration of PE to constrict the vessel to 50% of the total diameter significantly increased in the CAST + T SHR compared to CONT (P < 0.05). Comparisons of passive structural mechanics between SHR and WKY treatment groups indicated in SHR a significantly increased wall-to-lumen ratio and decreased circumferential wall stress compared to WKY treatment groups. CONCLUSIONS: These data suggest that T and NE are involved in a complex interaction with both myogenic reactivity and structural alterations of resistance-sized blood vessels and that these factors likely contribute to the development and maintenance of hypertension.

Angiogenic growth factors are new and essential players in the sustained relaxin vasodilatory pathway in rodents and humans.

Relaxin is emerging as an important vasodilator of pregnancy and is being tested for afterload reduction in acute heart failure. However, the mechanisms underlying relaxin-induced vasodilation are incompletely understood. The aims of this study were to establish a new in vitro model for relaxin-induced vasodilation and to use this approach, as well as chronically instrumented, conscious rats, to investigate the role of angiogenic growth factors in the relaxin vasodilatory pathway. Incubation of rat and mouse small renal arteries with recombinant human H2 relaxin for 3 hours in vitro attenuated myogenic constriction, which was blocked by inhibitors of gelatinases, the endothelin B receptor, and NO synthase. These findings corroborate ex vivo observations in arteries isolated from relaxin-infused nonpregnant and midterm pregnant rats, thereby validating the new experimental approach and enabling the study of human arteries. Incubation of small human subcutaneous arteries with relaxin for 3 hours in vitro also attenuated myogenic constriction through the same molecular intermediates. Vascular endothelial growth factor receptor inhibitor SU5416, 3 different vascular endothelial growth factor, and 2 different placental growth factor neutralizing antibodies prevented relaxin from attenuating myogenic constriction in rat and mouse small renal and human subcutaneous arteries. SU5416 administration also prevented relaxin-induced renal vasodilation and hyperfiltration in chronically instrumented, conscious rats. Small renal arteries isolated from these rats demonstrated increased matrix metalloproteinase 2 activity in the relaxin-infused group, which was not prevented by SU5416. We conclude that there is concordance of relaxin vasodilatory mechanisms in rats, mice, and humans, and angiogenic growth factors are novel and essential intermediates.

Relaxin induces rapid dilation of rodent small renal and human subcutaneous arteries via PI3 kinase and nitric oxide.

The peptide hormone relaxin is a potent vasodilator with therapeutic potential in diseases complicated by vasoconstriction, including heart failure. However, the molecular mediators and magnitude of vasodilation may vary according to duration of exposure and artery type. The objective of these studies was to determine mechanisms of rapid (within minutes) relaxin-induced vasodilation and to examine whether relaxin dilates arteries from different animal species and vascular beds. Rat and mouse small renal, rat mesenteric, and human sc arteries were isolated, mounted in a pressure arteriograph, and treated with recombinant human relaxin (rhRLX; 1-100 ng/ml) after preconstriction with phenylephrine. Rat and mouse small renal as well as human sc arteries dilated in response to rhRLX, whereas rat mesenteric arteries did not. Endothelial removal or pretreatment with l-N(G)-monomethyl arginine (L-NMMA) abolished rapid relaxin-induced vasodilation; phosphatidylinositol-3-kinase (PI3K) inhibitors also prevented it. In cultured human endothelial cells, rhRLX stimulated nitric oxide (assessed using 4-amino-5-methylamino-2'7'-difluorofluorescein) as well as Akt and endothelial NO synthase (eNOS) phosphorylation by Western blotting but not increases in intracellular calcium (evaluated by fura-2). NO production was attenuated by inhibition of Gα(i/o) and Akt (using pertussis toxin and the allosteric inhibitor MK-2206, respectively), PI3K, and NOS. Finally, the dilatory effect of rhRLX in rat small renal arteries was unexpectedly potentiated, rather than inhibited, by pretreatment with the vascular endothelial growth factor receptor inhibitor SU5416. We conclude that relaxin rapidly dilates select arteries across a range of species. The mechanism appears to involve endothelial Gα(i/o) protein coupling to PI3K, Akt, and eNOS but not vascular endothelial growth factor receptor transactivation or increased calcium.

Testosterone increases myogenic reactivity of second-order mesenteric arteries in both defective and normal androgen receptor adult male rats.

BACKGROUND: Testosterone (T) and the androgen receptor (AR) are involved in mechanisms associated with hypertension and vessel reactivity. OBJECTIVE: To investigate T and the AR on blood vessel reactivity, testicular feminized male (TFM; AR deficient males) and normal androgen receptor (NAR) male rats were used. Therefore, if the functional AR is necessary for plasma T to regulate vessel responsiveness, TFM males will exhibit altered vessel function compared to NAR males. METHODS: Adult (16 weeks of age) TFM or NAR males were assigned to the following treatment groups: gonadal intact controls (CONT), castrate (CAST), or castrate with T replacement (CAST+T) with (n=8-10/group). RESULTS: Plasma T followed a consistent pattern with CAST+T elevated compared to CONT and CAST TFM and NAR males. In addition, CAST plasma T was significantly decreased compared to CONT and CAST+T in TFM and NAR males. In a similar manner for systolic blood pressure (SBP), CAST lowered SBP compared to CONT in both NAR and TFM. Following 8 weeks of treatment, second-order mesenteric artery responses to changes in intraluminal pressure (myogenic reactivity) were analyzed using a pressure arteriograph system. Both TFM (P < 0.05) and NAR (P < 0.05) CAST groups revealed a decrease in myogenic reactivity compared to CONT. Following T treatment the TFM CAST+T myogenic reactivity returned to CONT levels, whereas the NAR CAST+T myogenic reactivity increased a further 10%. CONCLUSION: The results of this study indicate that T differentially regulates mesenteric artery reactivity in TFM and NAR males. Our data also demonstrate that both AR and/or non-AR mediated mechanisms may partially contribute to SBP regulation.

Hypertension induced by episodic reductions in uteroplacental blood flow in gravid rat.

BACKGROUND: The etiology of preeclampsia remains unclear. Animal modeling of preeclampsia has been useful; however, no model to date represents episodic changes in uteroplacental blood flow that may occur in preeclampsia. OBJECTIVE: To develop a gravid rat model characterized by episodic reductions in uteroplacental blood flow. METHOD: Pregnant Sprague Dawley rats were used and subjected to SHAM, reduced uterine perfusion pressure (RUPP), or aortic occlusion on gestational Day 14. Aortic occlusion surgery consisted of implantation of a silastic vascular occluder around the abdominal aorta and silver clips around the uterine-ovarian arteries. Aortic occlusion animals were subjected to five consecutive days of occlusion (40% reduction) each session lasting 1 h. On Day 21, maternal mean arterial pressure (MAP) and fetal morphology were assessed. For isolated blood vessels, resistance-sized mesenteric arteries were harvested and mounted on a pressure arteriograph. RESULT: Occluder animals experienced a 10 mmHg rise in MAP as compared to SHAM (p < 0.05), and RUPP MAP was significantly increased as compared to control subjects (p < 0.05). Pups from Occluder animals exhibited a decrease in fetal weight as compared to SHAM (p < 0.05), but an increase in fetal weight as compared to RUPP (p < 0.05). Myogenic reactivity of second-order mesenteric arteries increased in Occluder animals as compared to SHAM (p < 0.05), but were similar to that of RUPP. CONCLUSION: Episodic reductions in uteroplacental blood flow play a crucial role in the altered vascular reactivity seen in Occluder animals and may represent a new model to investigate the mechanisms associated with episodic reductions in uteroplacental blood flow in pathological pregnancies.

Increased myogenic responses of resistance-sized mesenteric arteries after reduced uterine perfusion pressure in pregnant rats.

OBJECTIVE: A central component of preeclampsia is a reduction in utero-placental perfusion. We tested the hypothesis that vascular reactivity of second - order mesenteric arteries would be increased in a pregnant rat with reduced uterine perfusion pressure (RUPP). METHODS: Pregnant 10-12 week old Sprague-Dawley rats underwent RUPP surgery on gestational day 14, and experiments were carried out on day 20. SHAM operated animals were used as controls. Resistance caliber mesenteric arteries (200-250 µm) were isolated, myogenic reactivity and responses to vasoconstrictor and vasodilator agonists were assessed utilizing a pressurized arteriograph system. RESULTS: RUPP resulted in maternal hypertension and reductions in fetal number and weight. Resistance caliber mesenteric arteries (200-250 µm) were isolated, myogenic reactivity and responses to vasoconstrictor and vasodilator agonists were assessed utilizing a pressurized arteriograph system. Myogenic reactivity responses were normalized as a percent change in vessel diameter from an initial diameter at 20 mmHg. The mesenteric arteries from RUPP animals exhibited a significant increase in myogenic reactivity compared to SHAM controls (p < 0.05). This increased myogenicity was reversed with prostaglandin inhibition, suggesting a role for a vasoconstrictor prostaglandin. In addition to alterations in myogenic reactivity, resistance-sized arteries from RUPP animals have decreased responses to nitric oxide (NO) as evidenced by decreases in responses to methacholine (ME; P < 0.05) and no change in myogenic reactivity after NO synthase blockade. CONCLUSION: RUPP alters the behavior of resistance caliber arteries to favor a more contractile phenotype with decreased in NO responses, which is similar to what is seen in preeclampsia.

Colony social stress differentially alters blood pressure and resistance-sized mesenteric artery reactivity in SHR/y and WKY male rats.

Increased sympathetic nervous system (SNS) activity, testosterone, and spontaneously hypertensive rat Y chromosome (SHR Yc) play a role in a genetic model of hypertension. Male rats with the SHR Yc and Wistar-Kyoto (WKY) autosomes (denoted SHR/y) exhibit these characteristics when compared to rats with the WKY Yc and WKY autosomes (denoted WKY). We hypothesized that chronic social stress will increase blood pressure and SNS activity more in SHR/y males compared to WKY males, resulting in increased myogenic reactivity along with decreased vasoconstriction of small mesenteric arteries. SHR/y and WKY males were housed in strain- specific colonies (10 males with 10 females) or as controls (10 males). Systolic blood pressure (SBP) and blood samples were collected prior to termination. Second-order mesenteric arteries were studied using a pressure arteriograph in which myogenic reactivity and phenylephrine (PE) responsiveness were measured. SHR/y colony SBP, and circulating norepinephrine and testosterone concentrations were elevated compared to control and WKY colony males (p < 0.05). Mesenteric artery myogenic reactivity was increased in SHR/y colony males (p < 0.001). Mesenteric arteries from SHR/y colony males exhibited a significant decrease in PE-induced constriction. Colony social stress elevated both SNS activity and testosterone level which may be responsible for the increased mesenteric artery myogenic reactivity, and SBP as noted in SHR/y males.

Vascular matrix metalloproteinase-9 mediates the inhibition of myogenic reactivity in small arteries isolated from rats after short-term administration of relaxin.

UNLABELLED: During pregnancy and chronic relaxin administration to nonpregnant rats (for days), vascular MMP (matrix metalloproteinase)-2 is increased and mediates renal vasodilation, hyperfiltration, and inhibition of myogenic reactivity of small renal arteries. However, the renal vasodilatory actions of relaxin also occur after only several hours of hormone administration to nonpregnant rats, and we hypothesized a pivotal role for vascular MMP-2. Accordingly, we used gelatin zymography, which reveals not only vascular MMP-2, but also MMP-9 activity in small renal arteries isolated from rats administered recombinant human relaxin (rhRLX) or vehicle for 4-6 h. Furthermore, we tested whether myogenic reactivity is inhibited, and if so, whether the inhibition is mediated by increased vascular MMP-2. Surprisingly, we detected no significant difference in either pro or active MMP-2 in small renal arteries isolated from rhRLX and vehicle control treatment groups. In contrast, vascular MMP-9 was up-regulated by 70% (P < 0.0005 vs. vehicle). These results were completely unexpected and novel. MMP-9 protein expression was confined to the vascular smooth muscle. MMP-9, but not MMP-2 activity, was also increased in mesenteric arteries after short-term rhRLX administration (P < 0.005 and >0.05 vs. vehicle, respectively). Myogenic reactivity was inhibited in small renal arteries isolated from nonpregnant rats treated with rhRLX for 4-6 h (P < 0.01 vs. vehicle) and was completely restored by incubation with MMP-9, but not MMP-2 neutralizing antibodies in vitro. CONCLUSION: In contrast to chronic rhRLX administration, MMP-9 rather than MMP-2 plays a central role in the vasodilatory effect of short-term relaxin administration.

Evidence for local relaxin ligand-receptor expression and function in arteries.

Relaxin is a 6 kDa protein hormone produced by the corpus luteum and secreted into the blood during pregnancy in rodents and humans. Growing evidence indicates that circulating relaxin causes vasodilatation and increases in arterial compliance, which may be among its most important actions during pregnancy. Here we investigated whether there is local expression and function of relaxin and relaxin receptor in arteries of nonpregnant females and males. Relaxin-1 and its major receptor, Lgr7, mRNA are expressed in thoracic aortas, small renal and mesenteric arteries from mice and rats of both sexes, as well as in small renal arteries from female tammar wallabies (an Australian marsupial). Using available antibodies for rat and mouse Lgr7 receptor and rat relaxin, we also identified protein expression in arteries. Small renal arteries isolated from relaxin-1 gene-deficient mice demonstrate enhanced myogenic reactivity and decreased passive compliance relative to wild-type (WT) and heterozygous mice. Taken together, these findings reveal an arterial-derived, relaxin ligand-receptor system that acts locally to regulate arterial function.

Increased arterial load alters aortic structural and functional properties during embryogenesis.

As in the adult dorsal aorta, the embryonic dorsal aorta is an important determinant of cardiovascular function, and increased stiffness may have secondary effects on cardiac and microcirculatory development. We previously showed that acutely and chronically increased arterial load via vitelline artery ligation (VAL) increases systemic arterial stiffness. To test the hypothesis that local dorsal aortic stiffness also increases, we measured aortic pulse-wave velocity (PWV) and assessed the active and passive properties (stress and strain) of isolated aortic segments. PWV along the dorsal aorta increased acutely and chronically after VAL. Analysis of isolated aortic active properties suggests that load-exposed aortas experienced higher stress, but not strain, at similar intraluminal pressures. When smooth muscle tone was relaxed, strain decreased in VAL vessels, whereas stress became similar to control vessels. Immunohistochemical analysis revealed that although aortic smooth muscle alpha-actin content was similar between groups, more cell layers expressed smooth muscle alpha-actin, and myocyte cell shape was markedly rounder in VAL embryos. Additionally, aortic and perivascular collagen type I and III content significantly increased in load-exposed VAL vessels. Increased production of these proteins is consistent with the observed increase in aortic PWV and decreased strain in VAL passive aortic segments. Thus the embryonic dorsal aorta is sensitive to increased arterial load and adapts by altering its material properties via changes in collagen content.

Moderate ascorbate deficiency increases myogenic tone of arteries from pregnant but not virgin ascorbate-dependent rats.

Plasma ascorbic acid is decreased in women with the pregnancy disorder preeclampsia. We used a mutant strain of rats (Osteogenic Disorder Shionogi), dependent on dietary sources of vitamin C, to investigate whether reduced intake of the vitamin would differentially affect vascular function in late-pregnant (day 19) and age-matched virgin rats. The animals were given either 1 mg/mL of ascorbic acid ad libitum in drinking water [fully supplemented (FS)] or 0.25 mg/mL [marginally supplemented (MS)]. Fetal weights were 21% lower in MS than FS rats, whereas mean maternal weights and pup numbers did not differ. Small mesenteric arteries (diameter, 268+/-7 microm) were mounted in a pressurized arteriograph. Myogenic reactivity (contractile response to step increases in intraluminal pressure) was increased in arteries from MS pregnant compared with FS pregnant rats to levels observed in virgin rats. Ascorbic acid intake did not affect myogenic responses of arteries from virgin rats. Hence, the normal pregnancy-induced reduction in myogenic reactivity was abrogated in MS pregnant animals. Inhibition of nitric oxide synthase had no effect on the myogenicity of arteries from virgin or MS pregnant rats but increased myogenicity of FS pregnant rats to the level of MS pregnant rats. Free radical scavengers reversed the accentuated myogenicity of MS pregnant rats without affecting FS pregnant or virgin rat arteries. These data indicate that moderate ascorbate deprivation increases mesenteric artery myogenic responsiveness during pregnancy. This increase may result from a decrease in nitric oxide-mediated modulation of the myogenic contractile response.

Role of relaxin in maternal renal vasodilation of pregnancy.

The remarkable hemodynamic changes of normal pregnancy are briefly reviewed. In addition, new findings and current concepts related to the underlying hormonal and molecular mechanisms are presented. Finally, work that is in progress as well as future directions is briefly discussed.

Evidence against the hypothesis that endothelial endothelin B receptor expression is regulated by relaxin and pregnancy.

The endothelial endothelin B (ET(B)) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ET(B) receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 mug/h or vehicle for 5 d or 4-6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ET(B) expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ET(B) protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET(B) expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET(B) expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ET(B) receptor protein has little or no role in relaxin stimulation of the endothelial ET(B)/nitric oxide vasodilatory pathway.

Emerging role of relaxin in renal and cardiovascular function.

Although traditionally associated with reproductive processes, relaxin is emerging as an important player in renal and cardiovascular function. Much of our recently acquired understanding of relaxin in this new context has arisen from studies of maternal renal and cardiovascular adaptations to pregnancy in rats where the hormone is turning out to be an important mediator. First, we highlight the influence of relaxin on renal hemodynamics and glomerular filtration rate, as well as on other peripheral circulations. Second, we discuss the effect of relaxin on both the steady and pulsatile systemic arterial load, as well as on the heart, in particular, coronary blood flow. Third, we consider the impact of the hormone on cultured endothelial and vascular smooth muscle cells. Fourth, we address the interaction of relaxin with renal and cardiac disease, as well as its role in angiogenesis. Finally, in Perspectives, we point out several key research questions in need of investigation that relate to a potential autocrine/paracrine role of relaxin in renal and cardiovascular tissues. Furthermore, on the basis of its potent vasodilatory and matrix-degrading attributes, we speculate about the therapeutic potential of relaxin in renal and cardiovascular diseases.

Nitric oxide synthase isoforms in the rat kidney during pregnancy.

OBJECTIVE: Nitric oxide mediates renal vasodilation and hyperfiltration during pregnancy in conscious rats through the endothelin B (ETB) receptor subtype. We tested the hypothesis that immunoreactive levels of endothelial nitric oxide synthase (eNOS) would be greater in the kidneys of midterm pregnant rats compared with virgin rats. METHODS: We studied midterm pregnancy because renal plasma flow and glomerular filtration rate are maximal at this gestational stage. Western analysis was used to determine the level of eNOS in the three major zones of the kidney-inner medulla, outer medulla, and cortex-and in isolated small renal arteries, and in purified renal microvessels from the cortex. RESULTS: There were no significant differences in eNOS expression between virgin and midterm pregnant rats in any of those renal tissues, regardless of whether immunoreactivity was expressed as arbitrary densitometry units, as "microg placental equivalents" interpolated from the linear portion of a dose-response curve of placental villous protein (2.5-30 microg, positive control) run concurrently on each gel, or normalized for beta-actin. We also investigated other NOS isoforms. In particular, immunoreactive neuronal NOS (nNOS) was detectable in the inner and outer medulla, but it was not significantly different between groups. nNOS immunoreactivity was below the level of detection in the cortex, but mRNA expression was not significantly different between pregnant and virgin rats by reverse transcriptase polymerase chain reaction. CONCLUSION: Our results suggest that an increase in eNOS isoform mass does not contribute to the endothelin and NO-dependent renal vasodilation in rat gestation.

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.

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.