Alie Kanu

Inhibition of leptin release by atrial natriuretic peptide (ANP) in human adipocytes

The addition of atrial natriuretic peptide (ANP) to isolated human adipocytes in primary culture from very obese individuals resulted in an inhibition of leptin release after a 24- or 48-hr incubation. There was also an inhibition of leptin release by isoproterenol (ISO) that was partially reversed by insulin, whereas the inhibition due to ANP was unaffected. Similar results were seen with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89), which is a cell-permeable inhibitor of protein kinase A. H-89 markedly reduced the effects of ISO on both lipolysis and leptin release without affecting the stimulation of lipolysis or the inhibition of leptin release due to ANP. Inhibition of endogenous nitric oxide formation using N(omega)-nitro-L-arginine resulted in a 20% increase in leptin release over 48 hr, which suggests that the nitric oxide/cyclic GMP pathway might play a small role in the regulation of endogenous leptin release. Similarly, the addition of the nitric oxide donor (Z)-1-[2-aminoethyl)-N-(2-aminoethyl)diazen-1-ium-1,2-diolate (DETA NONOate) at 0.1 or 1 microM to explants of human adipose tissue enhanced lipolysis by 29%. Our data demonstrate that the lipolytic effect of ANP is probably secondary to stimulation of cyclic GMP accumulation in human adipocytes, and this is accompanied by an inhibition of leptin release.

Roles of Glia Limitans Astrocytes and Carbon Monoxide in Adenosine Diphosphate-Induced Pial Arteriolar Dilation in Newborn Pigs

Background and Purpose— Astrocytes, neurons, and microvessels together form a neurovascular unit allowing blood flow to match neuronal activity. Adenosine diphosphate (ADP) is an important signaling molecule in the brain, and dilation in response to ADP is astrocyte-dependent in rats and newborn pigs. Carbon monoxide (CO), produced endogenously by catabolism of heme to CO, iron, and biliverdin via heme oxygenase, is an important cell-signaling molecule in the neonatal cerebral circulation. We hypothesize ADP stimulates CO production by glia limitans astrocytes and that this CO causes pial arteriolar dilation. Methods— Experiments were performed using anesthetized piglet with closed cranial windows, and freshly isolated piglet astrocytes and microvessels. Astrocyte injury was caused by topical application of L-2-alpha aminoadipic acid (2 mmol/L, 5 hours). Cerebrospinal fluid was collected from under the cranial windows for measurement of ADP-stimulated CO production. CO was measured by gas chromatography–mass spectroscopy analysis. Results— Before, but not after, astrocyte injury in vivo, topical ADP stimulated both CO production and dilation of pial arterioles. Astrocyte injury did not block dilation to isoproterenol or bradykinin. Chromium mesoporphyrin, an inhibitor of heme oxygenase, also prevented the ADP-induced increase in cerebrospinal fluid CO and pial arteriolar dilation caused by ADP, but not dilation to sodium nitroprusside. ADP also increased CO production by freshly isolated piglet astrocytes and cerebral microvessels, although the increase was smaller in the microvessels. Conclusions— These data suggest that glia limitans astrocytes use CO as a gasotransmitter to cause pial arteriolar dilation in response to ADP.

Cyclooxygenase products stimulate carbon monoxide production by piglet cerebral microvessels.

Products of arachidonic acid (AA) metabolism by cyclooxygenase (Cox) are important in regulation of neonatal cerebral circulation. The brain and cerebral microvessels also express heme oxygenase (HO) that metabolizes heme to carbon monoxide (CO), biliverdin, and iron. The purpose of this study in newborn pig cerebral microvessels was to address the hypothesis that Cox products affect HO activity and HO products affect Cox activity. AA (2.0–20 u.μM) increased prostaglandin E2 (PGE2) measured by radioimmunoassay (RIA) and also CO measured by gas chromatography/mass spectrometry (GC/MS). Further, 10-4 M indomethacin, which inhibited Cox, reduced both AA and heme-induced CO production. Conversely, neither exogenous 2 × 106 M heme, which markedly increased CO production, nor the inhibitor of HO, chromium mesoporphyrin, altered PGE2 synthesis. Because AA metabolism by Cox generates both prostanoids and superoxides, we determined the effects of the predominant prostanoid and superoxide on CO production. Although PGE2 caused a small increase in CO production, xanthine oxidase plus hypoxanthine, which produces superoxide, strongly stimulated the production of CO by cerebral microvessels. This increase was mildly attenuated by catalase. These data suggest that Cox-catalyzed AA metabolites, most likely superoxide and/or a subsequent reactive oxygen species, increase cerebrovascular CO production. This increase seems to be caused, at least in part, by the elevation of HO-2 catalytic activity. Conversely, Cox activity is not affected by HO-catalyzed heme metabolites. These data suggest that some cerebrovascular functions attributable to Cox activity could be mediated by CO.

Arachidonic acid- and prostaglandin E2-induced cerebral vasodilation is mediated by carbon monoxide, independent of reactive oxygen species in piglets

Arachidonic acid (AA) and prostaglandin (PG) E(2) stimulate carbon monoxide (CO) production, and AA metabolism is known to be associated with the generation of reactive oxygen species (ROS). This study was conducted to address the hypothesis that CO and/or ROS mediate cerebrovascular dilation in newborn pigs. Experiments were performed on anesthetized newborn pigs with closed cranial windows. Different concentrations of AA (10(-8)-10(-6) M), PGE(2) (10(-8)-10(-6) M), iloprost (10(-8)-10(-6) M), and their vehicle (artificial cerebrospinal fluid) were given. Piglets with PGE(2) and iloprost received indomethacin (5 mg/kg iv) to inhibit cyclooxygenase. AA, PGE(2), and iloprost caused concentration-dependent increases in pial arteriolar diameter. The effects of both AA and PGE(2) in producing cerebral vascular dilation and associated CO production were blocked by the heme oxygenase inhibitor chromium mesoporphyrin (2 × 10(-5) M), but not by the prostacyclin analog, iloprost. ROS inhibitor tempol (SOD mimetic) (1 × 10(-5) M) and the H(2)O(2) scavenger catalase (1,000 U/ml) also do not block these vasodilator effects of AA and PGE(2). Heme-L-lysinate-induced cerebrovascular dilation and CO production was blocked by chromium mesoporphyrin. Hypoxanthine plus xanthine oxidase, a combination that is known to generate ROS, caused pial arteriolar dilation and CO production that was inhibited by tempol and catalase. These data suggest that AA- and PGE(2)-induced cerebral vascular dilation is mediated by CO, independent of ROS.

Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice

Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg(-1)·min(-1)) infused by miniosmotic pumps for 2 wk in female Cyp1b1(+/+) mice did not alter water consumption, urine output, Na(+) excretion, osmolality, or protein excretion. However, in Cyp1b1(-/-) mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na(+) excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-β in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17β-estradiol metabolite 2-methoxyestradiol in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17β-estradiol.