Donald J. DiPette

Increased Blood Pressure in α-Calcitonin Gene–Related Peptide/Calcitonin Gene Knockout Mice

Abstract —Nerves that contain calcitonin gene–related peptide (CGRP) are components of the sensory nervous system. Although these afferent nerves have traditionally been thought to sense stimuli in the periphery and transmit the information centrally, they also have an efferent vasodilator function. Acute administration of a CGRP receptor antagonist increases the blood pressure (BP) in several models of hypertension, which indicates that this potent vasodilator plays a counterregulatory role to attenuate the BP increase in these settings. To determine the role of this peptide in the long-term regulation of cardiovascular function, including hypertension, we obtained mice that have a deletion of the α-calcitonin gene–related peptide (α- CGRP ) gene. Although the β-calcitonin gene–related peptide (β- CGRP ) gene is intact in these mice, α- CGRP is by far the predominant species of CGRP produced in dorsal root ganglia (DRG) sensory neurons. Initially, we examined the effect of deletion of the α- CGRP on baseline BP and β- CGRP and substance P mRNA expression. Systolic BP was significantly higher in the knockout mice (n=7) compared with wild-type in both male (160±6.1 vs 125±4.8 mm Hg) and female (163±4.8 vs 135±33 mm Hg) mice. Next, groups (n=7) of knockout and wild-type mice had catheters surgically placed in the right carotid artery for mean arterial pressure recording. With the animals fully awake and unrestrained, the knockout mice displayed an elevated mean arterial pressure compared with wild-type in both male (139±4.9 vs 118±4.9 mm Hg) and female (121±3.4 vs 107±3.1 mm Hg) mice. Northern blot analysis of DRG RNA samples confirmed the absence of α- CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, β- CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that α- CGRP may be involved in the long-term regulation of resting BP and suggest that these mice are particularly sensitive to challenges to BP homeostasis because of the loss of a compensatory vasodilator mechanism.

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH‐SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH‐SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH‐SY5Y cells in a transamidation‐independent manner. RA‐induced activation of Rac1 is mediated by phosphatidylinositol 3‐kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over‐expression of constitutively active PI3K or Rac1‐V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over‐expression of dominant negative Rac1‐N17, blocks RA‐induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH‐SY5Y cells.

Regulation of Calcitonin Gene-Related Peptide Expression in Dorsal Root Ganglia of Rats by Female Sex Steroid Hormones

Abstract Calcitonin gene-related peptide (CGRP), a potent vasodilator primarily synthesized in dorsal root ganglia (DRG) neurons, has been shown to decrease vascular resistance and thus regulate blood flow to a variety of organs in rats. Serum CGRP levels in the human have been reported to increase with pregnancy and decrease postpartum. It has been suggested that female sex steroid hormones play a role in cardiovascular function, but the mechanisms are unknown. In this study, we examined the effects of estradiol-17β (E2) and progesterone (P4) on the expression of CGRP in DRG in adult rats both in vivo and in vitro. Ovariectomized (ovx) animals were injected s.c. with 5 μg E2, 4 mg P4, or 5.0 μg E2 + 4 mg P4 in 0.5 ml sesame oil or with oil only, and groups of 4 rats were killed at 0, 24, or 48 h. DRGs were then removed and analyzed for CGRP mRNA and immunoreactive (i-)CGRP content by Northern blotting and RIA, respectively. Primary cultures of DRG neurons from adult female rats were used to assess the effects of varying doses of E2 (1, 10, 100 nM), P4 (10, 100, 1000 nM), or E2 (10 nM) + P4 (100 nM) in the absence or presence of nerve growth factor (NGF; 20 ng/ml); and CGRP mRNA content in the cells and i-CGRP in the medium were quantitated at 24 or 48 h after incubation. Results of in vivo studies showed that E2 caused a significant increase in CGRP mRNA at 24 h (1.8-fold) and in i-CGRP levels both at 24 h (2.8-fold) and at 48 h (3.4-fold) in DRG of ovx rats. P4 also stimulated expression of both CGRP mRNA and i-CGRP. In the in vitro studies, either E2 or P4 alone or the two in combination were without effect on CGRP expression in cultured DRG neurons at all the doses tested. However, in the presence of NGF, both CGRP mRNA and peptide levels were significantly enhanced by E2, P4, and E2+P4 in a time-dependent (2.0- to 2.8-fold at 24 h, 3.0- to 5.0-fold at 48 h) and dose-dependent manner, with maximal effects achieved at 1.0 nM (E2) and 100 nM (P4) at 24 h of incubation. In summary, both E2 and P4, either alone or in combination, stimulate CGRP peptide synthesis in DRG neurons through increasing CGRP mRNA. The effects of these steroid hormones are mediated through amplifying the NGF-induced synthesis of CGRP in these neurons. Thus, we propose that the cardiovascular functions of female sex steroid hormones may be mediated, at least in part, by the up-regulation of neuronal CGRP synthesis, via NGF-mediated mechanisms.

Dose-Dependent Systemic and Regional Hemodynamic Effects of Calcitonin Gene-Related Peptide

The dose-response effects of infused calcitonin gene-related peptide (CGRP), a potent vasodilator, on systemic and regional hemodynamics in the conscious rat remain incompletely defined. The radioactive microsphere technique provided these determinations before and after the intravenous administration of vehicle or 22, 65, 220, and 2200 pmol of CGRP. Neither vehicle nor 22 pmol of CGRP significantly changed any systemic or regional hemodynamic parameter. Starting at the 65-pmol dose, CGRP significantly decreased mean blood pressure and total peripheral resistance, while increasing heart rate without changing cardiac output. CGRP produced selective regional vasodilatory effects, with the coronary circulation being unusually sensitive. In contrast, CGRP caused significant increases in blood flow to the mesenteric and cutaneous circulations only at the two highest doses. CGRP increased plasma norepinephrine, epinephrine, and renin activity significantly at only the 2200-pmol dose. In conclusion, CGRP decreases blood pressure by peripheral vasodilation, with a threshold dose occurring between 22 and 65 pmol. In addition, the coronary circulation appears to be particularly sensitive to the vasodilatory properties of CGRP.

Calcitonin Gene–Related Peptide Is a Depressor of Deoxycorticosterone-Salt Hypertension in the Rat

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide. We previously demonstrated that neuronal CGRP expression is significantly increased in deoxycorticosterone (DOC)-salt hypertensive rats. To determine the hemodynamic role of CGRP in this setting, we used CGRP8-37, a specific CGRP receptor antagonist. DOC-salt hypertension was induced in Sprague-Dawley rats. To control for DOC pellet implantation, left nephrectomy, and/or saline drinking water, we also studied four normotensive groups. Four week after the initiation of each protocol, all rats had intravenous (for drug administration) and arterial (for continuous mean arterial pressure monitoring) catheters surgically placed and were studied in the conscious, unrestrained state. Baseline mean arterial pressure was higher in the DOC-salt than normotensive rats (175 +/- 5 versus 119 +/- 4 mm Hg, P < .001). Vehicle administration did not alter mean arterial pressure in any group, and CGRP8-37 administration (bolus doses of 3.2 x 10(4) or 6.4 x 10(4) pmol/L) did not change mean arterial pressure in the four normotensive groups. However, CGRP8-37 administration to the DOC-salt rats rapidly and significantly increased mean arterial pressure at both the lower dose (9 +/- 1 mm Hg, P < .001) and higher dose (14 +/- 1 mm Hg, P < .001). In addition, the increase in mean arterial pressure between the two CGRP8-37 doses was also significant (P < .01), indicating a dose-dependent response. We conclude that the increase in neuronal CGRP expression in DOC-salt hypertension plays a compensatory vasodilator role to attenuate the elevated blood pressure. These results provide the first conclusive evidence that CGRP plays a direct role in DOC-salt hypertension.

Resveratrol Restores Nrf2 Level and Prevents Ethanol-Induced Toxic Effects in the Cerebellum of a Rodent Model of Fetal Alcohol Spectrum Disorders

In humans, ethanol exposure during pregnancy produces a wide range of abnormalities in infants collectively known as fetal alcohol spectrum disorders (FASD). Neuronal malformations in FASD manifest as postnatal behavioral and functional disturbances. The cerebellum is particularly sensitive to ethanol during development. In a rodent model of FASD, high doses of ethanol (blood ethanol concentration 80 mM) induces neuronal cell death in the cerebellum. However, information on potential agent(s) that may protect the cerebellum against the toxic effects of ethanol is lacking. Growing evidence suggests that a polyphenolic compound, resveratrol, has antioxidant and neuroprotective properties. Here we studied whether resveratrol (3,5,4′-trihydroxy-trans-stilbene), a phytoalexin found in red grapes and blueberries, protects the cerebellar granule neurons against ethanol-induced cell death. In the present study, we showed that administration of resveratrol (100 mg/kg) to postnatal day 7 rat pups prevents ethanol-induced apoptosis by scavenging reactive oxygen species in the external granule layer of the cerebellum and increases the survival of cerebellar granule cells. It restores ethanol-induced changes in the level of transcription factor nuclear factor-erythroid derived 2-like 2 (nfe2l2, also known as Nrf2) in the nucleus. This in turn retains the expression and activity of its downstream gene targets such as NADPH quinine oxidoreductase 1 and superoxide dismutase in cerebellum of ethanol-exposed pups. These studies indicate that resveratrol exhibits neuroprotective effects in cerebellum by acting at redox regulating proteins in a rodent model of FASD.

Calcitonin Gene-Related Peptide Is a Depressor in NG-Nitro-l-Arginine Methyl Ester-Induced Hypertension During Pregnancy

Inhibition of nitric oxide production with NG-nitro-L-arginine methyl ester (L-NAME) increases blood pressure and fetal mortality in pregnant rats. We previously reported that administration of calcitonin gene-related peptide (CGRP) reduces the blood pressure and fetal death produced by L-NAME. To determine the hemodynamic role of endogenous CGRP in this setting, CGRP8-37, a CGRP receptor antagonist, was used. In addition, CGRP mRNA and peptide levels were determined in dorsal root ganglia. L-NAME or control rats had intravenous (for drug administration) and arterial (for continuous mean blood pressure monitoring) catheters surgically placed and were studied in the conscious unrestrained state. Baseline blood pressure was higher in the L-NAME than the control rats on days 19, 20, and 21 or pregnancy and postpartum day 1. Vehicle administration did not change blood pressure in any group, and CGRP8-37 (100 micrograms) did not change blood pressure in control groups. However, CGRP8-37 administration to the L-NAME rats further increased blood pressure (P < .05) on days 19 (8 +/- 1), 20 (12 +/- 2), and 21 (7 +/- 1) of gestation but was without effect on postpartum day 1. Furthermore, CGRP mRNA or peptide levels in dorsal root ganglia were not different between the L-NAME and control rats at any of the time points studied. These data indicate that in experimental preeclampsia, CGRP is playing a compensatory vasodilator role to attenuate the elevated blood pressure. The mechanism of this effect appears to be an enhanced vascular responsiveness to CGRP that is attenuated after the birth of pups.

Dexamethasone and activators of the protein kinase A and C signal transduction pathways regulate neuronal calcitonin gene-related peptide expression and release.

Primary cultures of adult rat dorsal root ganglia (DRG) neurons were used to determine if activation of either the protein kinase A or C signal transduction pathways or treatment with the synthetic glucocorticoid dexamethasone modulate neuronal calcitonin gene-related peptide (CGRP) synthesis and release. DRG are the sites of neuronal cell bodies known to produce abundant CGRP levels, and to send axons peripherally to blood vessels and centrally to the spinal cord. Using immunocytochemical techniques, we confirmed that synthesis of immunoreactive CGRP (iCGRP) is restricted to a subpopulation of DRG neurons. Subsequently, we determined that treatment (24 h) of the neurons with either dibutyryl cAMP (1 mM) or phorbol 12-myristate 13-acetate (2 microM) increased CGRP mRNA content 2.2 +/- 0.4 (n = 6, p < 0.03) and 3.0 +/- 0.6-fold (n = 6, P < 0.02) respectively, while secreted iCGRP levels were increased 1.8 +/- 0.2 (n = 14, P < 0.005) and 4.5 +/- 1.0 (n = 14, P < 0.001)-fold over control levels. Treatment of the neurons with dexamethasone alone had no effect on CGRP expression; however, this agent was able to significantly attenuate the stimulatory effects of NGF on both CGRP mRNA accumulation and release of iCGRP. Time course studies demonstrated that in the phorbol ester treated neurons CGRP mRNA levels continued to increase at 48 h, while maximal induction with dibutyryl cAMP occurred at approximately 12 h. These results indicate that local and/or circulating factors which act through the protein kinase A and C signal transduction pathways upregulate both CGRP expression and release, while glucocorticoids attenuate the stimulatory effects of NGF.

Resveratrol prevents embryonic oxidative stress and apoptosis associated with diabetic embryopathy, and improves glucose and lipid profile of diabetic dam

SCOPE Diabetic embryopathy, a consequence of diabetic pregnancy, is associated with increase in embryonic oxidative stress and apoptosis, which lead to severe embryonic damage at early stage of organogenesis. METHODS AND RESULTS This study investigated if resveratrol, found in red grapes and blue-berries, may prevent diabetes-induced oxidative stress and apoptosis in embryos and have beneficial effects in diabetic dams. A rodent model of diabetic embryopathy was used. Diabetes was associated with lowered reduced glutathione levels (26.98%), increased total thiol (100.47%) and lipid peroxidation (124.73%) in embryos, and increased blood sugar (384.03%), cholesterol (98.39%) and triglyceride (1025.35%) in diabetic dams. Increased apoptosis (272.20%) was also observed in the embryos of diabetic dams. Administration of resveratrol (100 mg/kg body weight (b.w.)) during pregnancy prevented both oxidative stress and apoptosis in embryos. Resveratrol reduced embryonic maldevelopment by improving embryo weight (41.23%), crown rump length (16.50%) and somite number (11.22%). It further improved the glucose (33.32%) and lipid (cholesterol 41.74%, triglyceride 60.64%) profile of the diabetic dams, which also represents the protective role of resveratrol in diabetes. CONCLUSION Resveratrol was found to prevent embryonic oxidative stress and apoptosis. It also improved glucose and lipid profile of diabetic dams, indicating the beneficial effects in diabetic pregnancy.

Calcitonin gene-related peptide gene expression in the spontaneously hypertensive rat.

Calcitonin gene-related peptide, a product of the calcitonin gene, is a potent vasodilator neuropeptide. We have demonstrated that dietary calcium deficiency decreased the neuronal (laminae I/II of the dorsal horn of the spinal cord) content of immunoreactive calcitonin gene-related peptide in the normal rat. Neuronal calcitonin gene-related peptide levels are also reduced in the spontaneously hypertensive rat, a model characterized by calcium deficiency. However, the mechanism of this reduction in neuronal calcitonin gene-related peptide could be due to decreased synthesis or increased release. To determine if neuronal calcitonin gene-related peptide messenger RNA (mRNA) levels are also decreased in the spontaneously hypertensive rat, we measured relative calcitonin gene-related peptide mRNA levels (using a genomic hybridization probe specific for alpha- and beta-calcitonin gene-related peptide mRNA) in dorsal root ganglia from spontaneously hypertensive and Wistar-Kyoto control rats. Dorsal root ganglia neuronal cell bodies are a prominent site of calcitonin gene-related peptide synthesis and send axons to peripheral blood vessels and central spinal cord sites (laminae I/II). After normalization of calcitonin gene-related peptide mRNA levels of 18S RNA, the calcitonin gene-related peptide mRNA/18S RNA ratio was significantly decreased approximately threefold in the spontaneously hypertensive rats compared with controls. This alteration in calcitonin gene-related peptide mRNA levels is specific for dorsal root ganglia, because no strain differences in calcitonin gene-related peptide mRNA content were detected in heart or brain.(ABSTRACT TRUNCATED AT 250 WORDS)