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Objective- The objective of this study was to investigate the effect of intravenous maternal sildenafil citrate (SC) administration on vascular function in growth-restricted fetal sheep. Approach and Results- Fetal growth restriction (FGR) results in cardiovascular adaptations that redistribute cardiac output to optimize suboptimal intrauterine conditions. These adaptations result in structural and functional cardiovascular changes, which may underlie postnatal neurological and cardiovascular sequelae. Evidence suggests SC, a potent vasodilator, may improve FGR. In contrast, recent clinical evidence suggests potential for adverse fetal consequence. Currently, there is limited data on SC effects in the developing fetus. We hypothesized that SC in utero would improve vascular development and function in an ovine model of FGR. Preterm lambs (0.6 gestation) underwent sterile surgery for single umbilical artery ligation or sham (control, appropriately grown) surgery to replicate FGR. Ewes received continuous intravenous SC (36 mg/24 h) or saline from surgery until 0.83 gestation. Fetuses were delivered and immediately euthanized for collection of femoral and middle cerebral artery vessels. Vessel function was assessed via in vitro wire myography. SC exacerbated growth restriction in growth-restricted fetuses and resulted in endothelial dysfunction in the cerebral and femoral vasculature, irrespective of growth status. Dysfunction in the cerebral circulation is endothelial, whereas smooth muscle in the periphery is the origin of the deficit. Conclusions- SC crosses the placenta and alters key fetal vascular development. Extensive studies are required to investigate the effects of SC on fetal development to address safety before additional use of SC as a treatment.

Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20-30 min. It has previously been shown that GTN induces headache in normal subjects after 20-30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms.

We have previously shown that atrial natriuretic peptide (ANP) is present in caveolae of in situ rat atrial myocytes. To investigate whether intracaveolar ANP of rat atrial myocytes exists within caveolae bound to type B ANP receptors (ANP-RB, a guanylyl cyclase), we have used confocal immunofluorescence microscopy applied to primary cultures of atrial myocytes from adult rats and to freshly dissociated rat atrial myocytes (not cultured). These experimental designs tested whether atrial myocyte ANP-RB colocalizes at the plasmalemma and elsewhere in the cell with the muscle-specific isoform of the caveolar coating protein caveolin-3, and with a fraction of cellular ANP. The experiments showed that cellular caveolin-3, a fraction of cellular ANP-RB, and a fraction of cellular ANP colocalize at the plasmalemma of cultured atrial myocytes and of freshly dissociated atrial myocytes. The observations support the hypothesis that in rat atrial myocytes, intracaveolar ANP is bound to ANP-RB, a protein whose cytosolic amino acid sequences are known to encode guanylyl cyclase activity. We suggest that among the (probably multiple) effects of the cGMP thus generated in the cytoplasmic microdomain underlying atrial myocyte caveolae may be the activation of cGMP-dependent protein kinase, which would thereby inhibit plasma membrane Ca2+ channel activity and contribute to a negative inotropic effect of ANP.

In vitro evidence suggests that natriuretic peptide receptors (NPR)-B and NPR-C inhibit vascular smooth muscle (VSM) proliferation. NPR-B is guanylate cyclase-coupled and selectively activated by C-type natriuretic peptide (CNP)-(1-22). NPR-C is not guanylate cyclase-coupled and, unlike NPR-B, avidly binds atrial natriuretic peptide (ANP)-(1-28) as well as CNP-(1-22). Here, we investigate these receptors during the VSM proliferation and neointimal formation found 5, 7, and 20 days after compressing the central ear artery of the rabbit. Receptors were mapped autoradiographically using [125I-Tyr0]CNP-(1-22), which binds NPR-B and NPR-C, and 125I-ANP-(1-28), which binds NPR-C and NPR-A, another guanylate cyclase-coupled receptor. Normal tunica media had NPR-B-like binding sites, and the level of these did not change significantly after compression. Consistent with this, CNP-(1-22) stimulated cGMP production equally with membranes from normal or damaged arteries and was more effective than ANP-(1-28). Neointima, which became evident 5 to 7 days after arterial damage, expressed NPR-C-like sites and no detectable NPR-B-like binding. NPR-C-like sites also appeared on the media for the first time between 5 and 7 days after compression. Immunohistochemistry for proliferating cell nuclear antigen revealed widespread mitosis in VSM at 5 days after compression, but mitosis was virtually restricted to the neointima at and beyond 7 days after compression. Thus, whereas levels of NPR-B did not change significantly after arterial injury and NPR-A was not detected, NPR-C-like receptors were upregulated as mitosis declined in the media and as a prominent neointima formed.

We investigated the vasoactive actions of the wound-healing agent tetrachlorodecaoxygen (TCDO). TCDO (20 microM) had no direct effect on tone in isolated calf pulmonary arteries precontracted with potassium with or without 1 microM reduced hemoglobin under O2 or N2 atmosphere. However, TCDO, in a reduced hemoglobin-dependent manner, attenuated contraction produced by serotonin, associated with spectral changes consistent with destruction of serotonin. The loss of tone induced by serotonin catalyzed by TCDO plus reduced hemoglobin was not altered in the presence of superoxide dismutase (SOD) plus catalase. TCDO plus reduced hemoglobin also produced rapid relaxation of isolated rabbit aorta precontracted with norepinephrine (NE), whereas with phenylephrine (PE)-induced bone, the observed relaxation was slow to develop. Neither did TCDO, with or without reduced hemoglobin, alter soluble guanylate cyclase activity in pulmonary artery. Thus, a highly reactive species produced by interaction of TCDO with reduced hemoglobin appears to attenuate the contractile actions of serotonin, NE, and PE, selectively potentially by destroying these vasoactive agents. The vasodilator actions of TCDO (plus reduced hemoglobin) may contribute to wound healing by increasing nutrient blood flow and O2 delivery needed for repair processes and bactericidal activity.

Cyclic nucleotides mediate the intracellular effects of various extracellular influences. To explore the possibility that nerve-muscle influences are mediated by cyclic nucleotides, we studied the effect of denervation on the cyclase enzymes of rabbit gastrocnemius using the contralateral, unoperated limb as the control. Adenylate cyclase activity decreased dramatically after denervation, while guanylate cyclase activity increased several times in all tissue fractions studied. Neither enzyme demonstrated cholinergic responsiveness. The dramatic changes in cyclase activities following denervation could result from a role of these enzymes in the mediation of nerve-muscle influences.

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.