Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes.

Prostaglandins (PGs) generated by the enzyme cyclooxygenase (COX) have been implicated in the pathological renal hemodynamics and structural alterations in diabetes mellitus, but the role of individual COX isoenzymes in diabetic nephropathy remains unknown. We explored COX-1 and COX-2 expression and hemodynamic responses to the COX-1 inhibitor valeryl salicylate (VS) or the COX-2 inhibitor NS398 in moderately hyperglycemic, streptozotocin-diabetic (D) and control (C) rats. Immunoreactive COX-2 was increased in D rats compared with C rats and normalized by improved glycemic control. Acute systemic administration of NS398 induced no significant changes in mean arterial pressure and renal plasma flow in either C or D rats but reduced glomerular filtration rate in D rats, resulting in a decrease in filtration fraction. VS had no effect on renal hemodynamics in D rats. Both inhibitors decreased urinary excretion of PGE(2). However, only NS398 reduced excretion of thromboxane A(2). In conclusion, we documented an increase in renal cortical COX-2 protein expression associated with a different renal hemodynamic response to selective systemic COX-2 inhibition in D as compared with C animals, indicating a role of COX-2-derived PG in pathological renal hemodynamic changes in diabetes.

Ovarian steroids and serotonin neural function.

The serotonin neural system originates from ten nuclei in the mid- and hindbrain regions. The cells of the rostral nuclei project to almost every area of the forebrain, including the hypothalamus, limbic regions, basal ganglia, thalamic nuclei, and cortex. The caudal nuclei project to the spinal cord and interact with numerous autonomic and sensory systems. This article reviews much of the available literature from basic research and relevant clinical research that indicates that ovarian steroid hormones, estrogens and progestins, affect the function of the serotonin neural system. Experimental results in nonhuman primates from this laboratory are contrasted with studies in rodents and humans. The sites of action of ovarian hormones on the serotonin neural system include effects within serotonin neurons as well as effects on serotonin afferent neurons and serotonin target neurons. Therefore, information on estrogen and progestin receptor-containing neurons was synthesized with information on serotonin afferent and efferent circuits. The ability of estrogens and progestins to alter the function of the serotonin neural system at various levels provides a cellular mechanism whereby ovarian hormones can impact mood, cognition, pain, and numerous other autonomic functions.

Lack of ovarian steroid hormone regulation of norepinephrine transporter mRNA expression in the non-human primate locus coeruleus.

Decreases in ovarian steroids can negatively affect mood, and drugs which block the norepinephrine transporter (NET) or the serotonin transporter (SERT) alleviate depression. However, the respective contribution of the noradrenergic and serotonergic systems may vary depending upon the etiology of the depression. We previously demonstrated that E and P alter gene expression for tryptophan hydroxylase (TPH) and for the serotonin reuptake transporter (SERT) in raphe neurons of the rhesus monkey. In this study, we questioned whether the noradrenergic system contributes to depression related to the reproductive function in women, using a non-human primate model of the menstrual cycle. The effect of estrogen (E) or E plus progesterone (P) on the expression of the NET gene in the locus coeruleus (LC) was examined with in situ hybridization for NET mRNA. In addition, we questioned whether the neurons of the LC contain nuclear E or P receptors (ER/PR). Hence, immunocytochemistry for ER and PR were performed on adjacent sections. Treatment groups consisted of monkeys (n = 4 per treatment) which were ovariectomized/hysterectomized (spayed), E-treated (28 days) and E+P-treated (14 days E, +14 days E+P). Expression of mRNA for NET was unchanged at any level of the LC due to steroid treatment (p > .05). Neither ER nor PR were detected in the LC of any treatment group. Therefore, E and P in a treatment paradigm which mimics the menstrual cycle do not directly regulate NET mRNA expression in the non-human primate LC. In addition, the noradrenergic neurons of the primate LC lack nuclear receptors for ovarian steroids. These data suggest that the noradrenergic system may not contribute significantly to depression related to changes in ovarian hormones.

Ovarian steroid effects on serotonin 1A, 2A and 2C receptor mRNA in macaque hypothalamus.

This study mapped the location of serotonin (5HT) 1A, 2A and 2C receptor mRNA expression in the female macaque hypothalamus and determined whether the expression was regulated by estrogen plus or minus progesterone treatment using in situ hybridization (ISH) and densitometric analysis of autoradiographic films. The experimental groups of pigtail macaques (Macaca nemestrina) were spayed controls (n=4), estrogen treated (28 days, n=4) and estrogen+progesterone-treated animals (14 days estrogen+14 days estrogen and progesterone, n=4). Monkey specific 5HT1A (432 bp), 2A (411 bp) and 2C (294 bp) receptor probes were generated with PCR. Moderate 5HT1A receptor hybridization signal was detected in the preoptic area and the ventromedial nuclei. Less intense 5HT1A receptor signal was detected in a contiguous area from the dorsomedial nuclei through the posterior hypothalamus and in the supramammillary area. There was no change in 5HT1A receptor hybridization signal in any area with ovarian steroid treatment. Dense 5HT2A receptor hybridization signal was morphologically confined to the paraventricular, supraoptic, and mammillary nuclei and the external capsule of the thalamus. Light 5HT2A mRNA signal was inconsistently observed in the ventromedial nuclei. There was no change in the 5HT2A receptor hybridization signal in any area with ovarian steroid treatment. The 5HT2C receptor mRNA was widely distributed in the macaque hypothalamus. The preoptic area and anterior hypothalamus were largely positive for 5HT2C mRNA with a more concentrated signal in a narrow periventricular area. Dense 5HT2C receptor signal was detected lateral to the ventromedial nuclei (capsule), in the tuberomammillary nuclei, arcuate nucleus, dorsomedial nuclei, infundibular area and choroid plexus. Moderate 5HT2C receptor signal was detected in the ventromedial nuclei, lateral hypothalamus and dorsal to posterior hypothalamus. There was a significant decrease in total 5HT2C mRNA hybridization signal with ovarian steroid treatment in the ventromedial nuclei, dorsal and posterior hypothalamus. In summary, macaque 5HT1A, 2A and 2C receptor mRNAs are located in distinct hypothalamic loci which play a role in a number of autonomic functions and behavior. Ovarian steroids decreased the expression of 5HT2C receptor mRNA in the ventromedial nuclei, dorsal and posterior hypothalamus. The expression of 5HT1A and 5HT2A receptor mRNA was not altered by treatment with ovarian steroids.

Impaired cholera toxin relaxation with age in rat aorta.

Beta-adrenergic-mediated vasorelaxation declines with maturation and aging. Available data suggest that impaired stimulatory G-protein function could explain this deficit. We have previously found a loss of cholera toxin (CT)-stimulated adenosine diphosphate (ADP) ribosylation with age in rat aortic membrane preparations, without evidence for loss of the stimulatory alpha subunit of G protein (Gsalpha) by immunoblotting. The purpose of this investigation was to determine if cholera toxin-mediated vasorelaxation was also impaired with age. Aortic ring segments from 6 weeks, 6 months, 12 months, and 24 months old male F-344 rats were used. Contraction to KCl and phenylephrine was assessed along with relaxation to cholera toxin (azide-free), isoproterenol and forskolin. There were no age-related changes to KCl or phenylephrine contraction. There was a significant decrease with age in relaxation to isoproterenol. This loss with age was significantly greater with KCl-preconstricted vessels than phenylephrine-preconstricted vessels. There were no age-related changes in the relaxation to forskolin. There was a significant decrease with age in the maximal relaxation to cholera toxin as well as a rightward shift in the dose-response curve. Cholera toxin-stimulated adenosine 3', 5'-cyclic phosphate (cAMP) levels were measured and there was no increase in cAMP levels surrounding the time period associated with relaxation induced by cholera toxin. These data suggest that different preconstricting agents markedly affect the age-related changes in beta-adrenergic-mediated vasorelaxation. Furthermore, they suggest that the mechanism of cholera toxin-mediated vasorelaxation may not be mediated through increases in cAMP concentration.

Steroid transformations in pregnant mares: metabolism of exogenous progestins and unusual metabolic activity in vivo and in vitro.

The mare possesses unique steroid hormone metabolic activity during pregnancy in that peripheral 4-pregnene-3,20-dione (progesterone; P4) is undetectable by 220 days gestation. This study examines in vivo metabolism of progestins by the pregnant mare and in vitro metabolic activity of maternal and fetal tissues. Pregnant mares (n = 3) received intravenous infusions of 3 beta-hydroxy-5-pregnen-20-one (pregnenolone; P5), P4, 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-5 alpha), deuterium labeled (D4)-P5, D4-3 beta-5 alpha and vehicle. Anestrous mares (n = 2) were infused with P5, P4, and vehicle. Also, placenta, endometrium, fetal gonad, maternal and fetal liver, and adrenal from 4 animals were incubated in D4-P5, D4-5 alpha-DHP, or D4-3 beta-5 alpha. Pregnant mares (in vivo) converted infused P5 predominantly to 5-pregnene-3 beta,20 beta-diol (P5-beta beta), 5 alpha-DHP, 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha-5 alpha), and 3 beta-5 alpha while only minor concentrations of P4 were detected. Infused P4 was converted primarily to 5 alpha-DHP and 20 alpha-hydroxypregnanes and when 5 alpha-DHP served as a substrate, other 5 alpha-pregnanes were formed. Infused 3 beta-5 alpha was either reduced to beta alpha-diol or oxidized to 5 alpha-DHP. Regardless of treatment, anestrous mares were incapable of producing any 20 alpha-hydroxypregnanes but could convert P5 to P5-beta beta and P4 in quantities similar to that of pregnant mares. In vitro, placenta converted D4-P5 to D4-P4 while D4-3 beta-5 alpha was oxidized to D4-5 alpha-DHP. Endometrium converted substrate primarily to D4-20 alpha-hydroxypregnanes. Both maternal and fetal liver produced D4-20 beta-hydroxy compounds regardless of substrate. Maternal and fetal adrenal were capable of conversion of D4-P5 to D4-P4 while fetal gonad did not perform any significant metabolism of substrate, though it produced P5. These data explain the absence of P4 and presence of other progestin metabolites in maternal circulation during mid- and late pregnancy. Pregnenolone can be 5 alpha-reduced to 3 beta-5 alpha, and 3 beta-5 alpha could be 3-oxidized to 5 alpha-DHP. It is 5 alpha-DHP that may serve as substrate for other 5 alpha-pregnanes.

Differential effect of trilostane on the progestin milieu in the pregnant mare.

Trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase, was administered intravenously to pregnant mares (n = 3) between day 277 and day 282 of gestation to determine its effect on the progestin milieu. In addition, placental tissue from mares at mid-gestation (150-300 days) (n = 4) were exposed to either deuterium-labelled pregnenolone alone or deuterium-labelled pregnenolone and trilostane to examine the effect of trilostane on placental metabolism of pregnenolone. Blood samples were collected from indwelling jugular catheters at frequent intervals for 48 h after infusion. Both plasma samples and incubation media were quantitatively analysed, after solid phase extraction and steroid derivitization, for concentrations of eight different progestin metabolites using gas chromatography and mass spectrometry. Trilostane infusion differentially affected the progestin milieu in vivo without inducing abortion. Forty-five minutes after infusion, maternal plasma concentrations of pregnenolone, 5-pregnene-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta,20 beta-diol and 3 beta-hydroxy-5 alpha-pregnan-20-one increased (P < 0.05) and remained high for 37 h. Concentrations of 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one and 5 alpha-pregnane-3 beta,20 alpha-diol decreased 15 min after infusion (P < 0.05), yet 1.5 h after infusion, concentrations had increased and remained high until 37 h after infusion. Trilostane inhibited the conversion of pregnenolone to progesterone in vitro (P < 0.001) while mediating an increase (P < 0.05) in concentrations of 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one. These observations demonstrate that the pregnant mare possesses unique steroid hormone metabolic activity and suggests that another steroid, perhaps 5 alpha-pregnane-3,20-dione and not progesterone, is the important steroid precursor for the other progestin metabolites found in circulating plasma.

Upregulation of G protein-linked receptor kinases with advancing age in rat aorta.

The age-related decline in beta-adrenergic receptor (beta-AR)-mediated vasorelaxation is associated with desensitization of beta-ARs without significant downregulation. The primary mode of this homologous beta-AR desensitization, in general, is via G protein receptor kinases (GRK). Therefore, we hypothesize that age-related changes in GRKs are causative to this etiology in rat aorta. Herein, we investigate the activity and cellular distribution (cytoplasmic vs. membrane) of several GRK isoforms and beta-arrestin proteins. GRK activity was assessed in extracts from aortic tissue of 6-wk, 6-mo, 12-mo, and 24-mo-old male Fischer-344 rats using a rhodopsin phosphorylation assay. We also performed immunoblots on lysates from aorta with specific antibodies to GRK-2, -3, -5, and beta-arrestin-1. Results show an age-related increase in GRK activity. Furthermore, expression of GRK-2 (cytoplasmic and membrane), GRK-3 (cytoplasmic and membrane), and beta-arrestin (soluble) increased with advancing age, whereas GRK-5 (membrane) expression remained unchanged. These results suggest that age is associated with increased activity and expression of specific GRKs. This increase likely results in enhanced phosphorylation and desensitization of beta-ARs. These biochemical changes are consistent with observed aging physiology.

Angiotensin II enhances beta-adrenergic receptor-mediated vasorelaxation in aortas from young but not old rats.

beta-Adrenergic receptor (beta-AR)-mediated (cAMP-dependent) vasorelaxation declines with advancing age. It has been shown that angiotensin II (ANG II), a potent vasoconstrictor, enhances cAMP-mediated vasorelaxation. Therefore, we questioned whether ANG II could reverse age-related, impaired beta-AR-mediated vasorelaxation and cAMP production. Pretreatment of aortic rings from 6-wk-old or 6-mo-old male Fischer 344 rats with ANG II significantly enhanced vasorelaxation induced by isoproterenol (Iso), a beta-AR agonist, and forskolin, a direct activator of adenylyl cyclase, but not dibutyryl-cAMP or isobutylmethylxanthine. The ANG II effect was blocked by losartan but not PD-123319 and was not observed in the aortas from 12- and 24-mo-old animals. Iso-stimulated cAMP production in the aorta was enhanced in the presence of ANG II in the 6-wk-old and 6-mo-old age groups only. Results suggest ANG II cannot reverse the age-related impairment in beta-AR-dependent vasorelaxation. We conclude aging may affect a factor common to both ANG II-receptors and beta-AR signaling pathways or aging may impair cross-talk between these two receptor pathways.

Viral-mediated gene delivery of constitutively activated G alpha s alters vasoreactivity.

1. Decline in beta-adrenoceptor (beta-AR)-mediated function occurs with increasing age, as well as in multiple disease conditions. The mechanisms responsible for this decline include alterations in beta-AR itself, beta-AR coupling proteins, such as G-proteins, or other beta-AR-linked proteins, such as G-protein receptor kinases and/or phosphatases. 2. The present study examines the physiological effects of in vitro transfer of constitutively activated G alpha s (G alpha s-Q227L) to both cultured vascular smooth muscle cells (VSMC) and whole aortic tissue of 6-month-old (adult) animals via a replication-deficient Herpes simplex virus (HSV) vector. These studies were conducted to provide a model for future examination of the role of G alpha s in the age-related decline in beta-AR-mediated vasorelaxation. 3. Gene transfer was confirmed by western blotting for specific proteins. Aortic tissue infected with HSV-G alpha s-Q227L had reduced phenylephrine-induced contraction and enhanced isoproterenol-stimulated vasorelaxation. Infection of cultured VSMC with HSV-G alpha s-Q227L increased both basal- and isoproterenol-stimulated cAMP accumulation, whereas forskolin-stimulated cAMP production was unchanged. 4. These results implicate G alpha s as a target for further investigation in age-related changes in vascular reactivity and support the use of viral-mediated gene transfer as an effective tool to study adrenergic signal transduction and physiology in vascular tissue.