Bradykinin and prostaglandin E1 regulate calcitonin gene-related peptide expression in cultured rat sensory neurons.

Primary cultures of adult rat dorsal root ganglia (DRG) sensory neurons were used to determine whether bradykinin and prostaglandins E1 (PGE1), E2 (PGE2) or I2 (PGI2) stimulate long-term calcitonin gene-related peptide (CGRP) mRNA accumulation and peptide release. Treatment (24 h) of neurons with either bradykinin or PGE1, significantly increased CGRP mRNA content and iCGRP release. However, PGE2 or PGI2 was without effect. Exposure of the cultured neurons to increasing concentrations of bradykinin or PGE1 demonstrated that the stimulation of CGRP expression was concentration-dependent, while time-course studies showed that maximal levels of CGRP mRNA accumulation and peptide release were maintained for at least 48 h. Treatment of the neuronal cultures with a bradykinin B2 receptor antagonist significantly inhibited the bradykinin-induced increase in CGRP expression and release. In addition, preincubation of neuronal cultures with the cyclooxygenase inhibitor indomethacin did not alter the PGE1-mediated stimulation of CGRP but blocked completely the bradykinin-induced increase in CGRP production. Therefore, these data indicate that bradykinin and PGE1 can regulate the synthesis and release of CGRP in DRG neurons and that the stimulatory effects of bradykinin on CGRP are mediated by a cyclooxygenase product(s). Thus, these findings suggest a direct relationship between chronic alterations in bradykinin/prostaglandin production that may arise from pathophysiological causes and long-term changes in CGRP expression.

Ethanol impairs Rho GTPase signaling and differentiation of cerebellar granule neurons in a rodent model of fetal alcohol syndrome.

Developmental exposure to ethanol impairs fetal brain development and causes fetal alcohol syndrome. Although the cerebellum is one of the most alcohol-sensitive brain areas, signaling mechanisms underlying the deleterious effects of ethanol on developing cerebellar granule neurons (CGNs) are largely unknown. Here we describe the effects of in vivo ethanol exposure on neurite formation in CGNs and on the activation of Rho GTPases (RhoA and Rac1), regulators of neurite formation. Exposure of 7-day-old rat pups to ethanol for 3 h moderately increased blood alcohol concentration (BAC) ( approximately 40 mM) and inhibited neurite formation and Rac1 activation in CGNs. Longer exposure to ethanol for 5 h resulted in higher BAC ( approximately 80 mM), induced apoptosis, inhibited Rac1, and activated RhoA. Studies demonstrated a regulatory role of Rho GTPases in differentiation of cerebellar neurons, and indicated that ethanol-associated impairment of Rho GTPase signaling might contribute to brain defects observed in fetal alcohol syndrome.

Retinoic acid receptors and tissue-transglutaminase mediate short-term effect of retinoic acid on migration and invasion of neuroblastoma SH-SY5Y cells.

Long-term treatment with all trans-retinoic acid (RA) induces neuronal differentiation and apoptosis. However, the effect of short-term RA treatment on cell proliferation, migration and invasion of neuroblastoma cell lines (SH-SY5Y and IMR-32) remains unclear. RA induces expression of tissue-transglutaminase (TGase) and promotes migration and invasion after 24 h of treatment in SH-SY5Y cells, but not in IMR-32 cells. RA receptor (RAR) agonist (4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid) and RAR/retinoid X receptor (RXR) agonist (9-cis-RA) promote expression of TGase, migration and invasion of SH-SY5Y cells, while RXR agonist has no significant effect. RAR antagonist blocks RA effect on migration and invasion, indicating that RAR receptors are required. Retinoid receptors are expressed and activated by RA in both cell lines. However, only transient activation of RAR is observed in IMR-32 cells. These findings suggest that different responses observed in SH-SY5Y and IMR-32 cells could be due to differential activation of retinoid receptors. Overexpression of TGase has no effect on migration or invasion, while overexpression of antisense TGase blocks RA-induced migration and invasion, indicating that other molecules along with TGase mediate RA effects. In addition to the long-term effects of RA that are coupled with cell differentiation, short-term effects involve migration and invasion of neuroblastoma SH-SY5Y cells.

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats.

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.

Role of sensory nervous system vasoactive peptides in hypertension.

The goal of the present research was to elucidate the roles and mechanisms by which the sensory nervous system, through the actions of potent vasodilator neuropeptides, regulates cardiovascular function in both the normal state and in the pathophysiology of hypertension. The animal models of acquired hypertension studied were deoxycorticosterone-salt (DOC-salt), subtotal nephrectomy-salt (SN-salt), and Nomega-nitro-L-arginine methyl ester (L-NAME)-induced hypertension during pregnancy in rats. The genetic model was the spontaneously hypertensive rat (SHR). Calcitonin gene-related peptide (CGRP) and substance P (SP) are potent vasodilating neuropeptides. In the acquired models of hypertension, CGRP and SP play compensatory roles to buffer the blood pressure (BP) increase. Their synthesis and release are increased in the DOC-salt model but not in the SN-salt model. This suggests that the mechanism by which both models lower BP in SN-salt rats is by increased vascular sensitivity. CGRP functions in a similar manner in the L-NAME model. In the SHR, synthesis of CGRP and SP is decreased. This could contribute to the BP elevation in this model. The CGRP gene knockout mouse has increased baseline mean arterial pressure. The long-term synthesis and release of CGRP is increased by nerve growth factor, bradykinin, and prostaglandins and is decreased by alpha2-adrenoreceptor agonists and glucocorticoids. In several animal models, sensory nervous system vasoactive peptides play a role in chronic BP elevation. In the acquired models, they play a compensatory role. In the genetic model, their decreased levels may contribute to the elevated BP. The roles of CGRP and SP in human hypertension are yet to be clarified.

Role of sensory nervous system vasoactive peptides in hypertension

The goal of the present research was to elucidate the roles and mechanisms by which the sensory nervous system, through the actions of potent vasodilator neuropeptides, regulates cardiovascular function in both the normal state and in the pathophysiology of hypertension. The animal models of acquired hypertension studied were deoxycorticosterone-salt (DOC-salt), subtotal nephrectomy-salt (SN-salt), and Nomega-nitro-L-arginine methyl ester (L-NAME)-induced hypertension during pregnancy in rats. The genetic model was the spontaneously hypertensive rat (SHR). Calcitonin gene-related peptide (CGRP) and substance P (SP) are potent vasodilating neuropeptides. In the acquired models of hypertension, CGRP and SP play compensatory roles to buffer the blood pressure (BP) increase. Their synthesis and release are increased in the DOC-salt model but not in the SN-salt model. This suggests that the mechanism by which both models lower BP in SN-salt rats is by increased vascular sensitivity. CGRP functions in a similar manner in the L-NAME model. In the SHR, synthesis of CGRP and SP is decreased. This could contribute to the BP elevation in this model. The CGRP gene knockout mouse has increased baseline mean arterial pressure. The long-term synthesis and release of CGRP is increased by nerve growth factor, bradykinin, and prostaglandins and is decreased by alpha2-adrenoreceptor agonists and glucocorticoids. In several animal models, sensory nervous system vasoactive peptides play a role in chronic BP elevation. In the acquired models, they play a compensatory role. In the genetic model, their decreased levels may contribute to the elevated BP. The roles of CGRP and SP in human hypertension are yet to be clarified

Role of calcitonin gene-related peptide and substance P in Dahl-salt hypertension.

Calcitonin gene-related peptide (CGRP) and substance P are known to play a counterregulatory role in acquired models of salt-dependent hypertension. In contrast, neuronal production of these peptides is decreased in the spontaneously hypertensive rat, which may contribute to the elevated blood pressure. To determine the role played by CGRP and substance P in Dahl-salt hypertension, 4- to 6-week-old male salt-resistant (DR) and salt-sensitive (DS) rats were divided into 4 groups (n=5/group) and pair-fed low-salt (0.2% NaCl) (DR/LS and DS/LS) and high-salt (8% NaCl) diets (DR/HS and DS/HS) for 3 weeks. After 3 weeks, all the rats had venous (for drug administration) and arterial (for blood pressure monitoring) catheters surgically implanted and were studied in the conscious and unrestrained state. Mean arterial pressure was significantly higher in the DS/HS rats animals (185.8+/-1.6 mm Hg, P<0.001). Intravenous administration of CGRP and SP receptor antagonists was without effect in any of the groups studied. CGRP and SP mRNA content from dorsal root ganglia were not significantly different between the groups. Whereas immunoreactive CGRP was decreased in the DS groups (DS/HS, 9.4+/-0.4 pg/microgram protein; DS/LS, 11.1+/-0.8 pg/microgram protein; P<0.01) compared with the DR groups (DR/HS, 13.9+/-0.6 pg/microgram protein; DR/LS, 14.6+/-0.6 pg/microgram protein), neuronal SP production was similar between all the groups. Thus, CGRP and substance P do not play a counterregulatory role in Dahl-salt hypertension. The decrease in neuronal CGRP expression in DS rats appears to be genetically determined as in SHR, however, and may contribute to the increase in blood pressure following salt-loading.

Omapatrilat in subtotal nephrectomy-salt hypertension: role of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide, plays a counterregulatory role in subtotal nephrectomy-salt (SN-salt)-induced hypertension, reflecting a stimulation of the efferent vasodilator function of perivascular sensory nerves. To determine the effect of omapatrilat, a dual ACE and neutral endopeptidase inhibitor, on blood pressure and the potential antihypertensive role for CGRP, 24 male Sprague-Dawley rats were separated into 4 groups: (1) SN-salt, (2) SN-salt plus omapatrilat (80 mg. kg(-1). d(-1) in the drinking water), (3) sham-operated plus salt, (4) sham-operated plus salt and omapatrilat. After 11 days the mean arterial pressure was higher in the SN-salt group (174+/-10 mm Hg) versus the sham-operated-salt (109+/-4 mm Hg) and sham-operated-salt plus omapatrilat (105+/-3 mm Hg) groups. Omapatrilat treatment of the SN-salt rats significantly decreased the mean arterial pressure to 123+/-7 mm Hg and significantly reduced the heart-to-body weight ratio. Intravenous administration of a specific CGRP receptor antagonist produced a significant 10+/-2 mm Hg mean arterial pressure increase in the untreated SN-salt hypertensive rats but was without effect in the other groups. This indicates that CGRP does not contribute to the antihypertensive actions of omapatrilat. In addition, CGRP mRNA and protein content in dorsal root ganglia were decreased approximately 25% in the SN-salt plus omapatrilat rats. Thus, omapatrilat not only markedly reduces the blood pressure in this model of renal failure-induced hypertension but may also prevent the abnormal compensatory stimulation of the vasodilator activity of the peripheral sensory nervous system.

Pregnancy and steroid hormones enhance the systemic and regional hemodynamic effects of calcitonin gene-related peptide in rats.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide known to be involved in the regulation of vascular resistance. Several lines of evidence suggest that CGRP plays a role in the vascular adaptations that occur during normal pregnancy; however, the effects of exogenous CGRP on systemic and regional hemodynamics during pregnancy remain unknown. Therefore, the purpose of this study was to determine the hemodynamic effects of systemically administered CGRP in adult pregnant (Day 19) and ovariectomized (ovx) rats using the radioactive microsphere technique. In addition, we also used ovariectomized rats treated for 3 days with estradiol (E2), progesterone (P4), E2 + P4 in sesame oil, or oil only to assess if these hormones regulate the CGRP-induced hemodynamic changes. On the day of study, catheters were inserted into the left cardiac ventricle (through the right carotid artery), right jugular vein, and caudal tail artery. Hemodynamic studies using radioactive microspheres were then performed in conscious rats 3 h after recovery from anesthesia. Blood pressure and heart rate were continuously monitored, and left ventricular pressure was determined immediately prior to each microsphere injection. Microspheres labeled with either (141)Ce or (85)Sr were injected prior to and 2 min following the i.v. bolus injection of CGRP (270 pmol/kg body weight [BW]). Mean arterial pressure (MAP) and total vascular resistance in pregnant rats was lower than in ovx rats, and this was further decreased with an i.v. bolus injection of 270 pmol CGRP/kg BW. Cardiac output was elevated with further increases upon CGRP administration in pregnant but not in ovx rats. The CGRP-induced changes in MAP, total vascular resistance, and cardiac output in E2 + P4 -treated rats were similar to that observed in Day 19 pregnant rats, indicating that CGRP effects on these parameters during pregnancy may be modulated by steroid hormones. Both pregnancy and E2 + P4 treatment in ovx rats caused significant decreases in CGRP-induced resistance in mesenteric, coronary, and renal vasculature. Thus, the vasodilatory sensitivity to CGRP during pregnancy may be mediated through decreased total vascular resistance, particularly to coronary, mesenteric, and renal vascular beds. Thus, CGRP-induced vasodilatory effects may play a role in mediating vascular adaptations that occur during pregnancy and that steroid hormones may modulate these CGRP effects.

Nerve growth factor enhances calcitonin gene-related peptide expression in the spontaneously hypertensive rat.

Calcitonin gene-related peptide (CGRP) expression is markedly reduced in dorsal root ganglia neurons in the spontaneously hypertensive rat (SHR). This decrease in such a potent vasodilator may contribute to the elevated blood pressure. Therefore, the purpose of this study was to determine whether stimulation of neuronal CGRP expression in SHR, by means of the administration of nerve growth factor, would lower the blood pressure. Nerve growth factor (10 nmol/L per kg/d, IP) was given to 12-week SHR (n=8 to 11/group) once a day for 1, 3, and 7 days. Control SHR received vehicle only. All rats were instrumented for CGRP receptor antagonist (CGRP(8-37)) administration (intravenous) and mean arterial pressure recording. Both the 1- and 3-day NGF treatments lowered the mean arterial pressure to 147+/-5 and 147+/-3 mm Hg, respectively, compared with controls (166+/-3 mm Hg). However, by day 7, the mean arterial pressure had returned to control levels (169+/-5 mm Hg). CGRP(8-37) administration produced a significant mean arterial pressure increase in all 3 nerve growth factor-treated groups (14+/-2, 10+/-2, and 13+/-2 mm Hg). CGRP mRNA levels in dorsal root ganglia were increased in the 3 neurotrophin-treated groups, whereas CGRP peptide content was higher at days 3 and 7. Therefore, nerve growth factor treatment of SHR can enhance neuronal CGRP expression. At days 1 and 3, nerve growth factor produces a depressor response that is primarily mediated by CGRP as evidenced by the pressor effect of CGRP(8-37.) At day 7, CGRP also plays a counterregulatory role, even though the mean arterial pressure has returned to control levels. This finding may result from a nerve growth factor-mediated upregulation of a pressor system that counteracts the hypotensive actions of CGRP. Thus, these data suggest that the decreased production of CGRP in SHR could contribute to the hypertension.

Use of the Ribonuclease Protection Assay for the Analysis and Characterization of Target mRNAs.

Some of the most widely used techniques in the area of molecular biology involve the isolation, analysis, and quantification of RNA molecules, specifically mRNA molecules that code for proteins of interest. Indeed, the characterization of any gene entails the analysis of the spatial and temporal distribution of RNA expression. In many types of studies, it is also necessary to quantify alterations in the synthesis of specific mRNA species that occur both under normal physiological conditions and in the pathophysiology of diseases such as hypertension. To date, the three most popular methods to characterize RNA molecules and determine the abundance of a particular mRNA in a total or poly (A) sample are Northern-blot analysis, ribonuclease protection assays (RPAs), and reverse transcription-polymerase chain reaction (RT-PCR). In theory, each of these techniques can be used to quantify either the relative or absolute level of an individual RNA species in a population. However, in practice, each method has inherent technical and practical limitations that may pose significant problems under certain circumstances (1,2).

Regulation of calcitonin gene-related peptide expression in dorsal root ganglia of rats by female sex steroid hormones.

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-17beta (E(2)) and progesterone (P(4)) 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 microg E(2), 4 mg P(4), or 5.0 microg E(2) + 4 mg P(4) 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 E(2) (1, 10, 100 nM), P(4) (10, 100, 1000 nM), or E(2) (10 nM) + P(4) (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 E(2) 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. P(4) also stimulated expression of both CGRP mRNA and i-CGRP. In the in vitro studies, either E(2) or P(4) 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 E(2), P(4), and E(2)+P(4) 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 (E(2)) and 100 nM (P(4)) at 24 h of incubation. In summary, both E(2) and P(4), 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.

Increased blood pressure in alpha-calcitonin gene-related peptide/calcitonin gene knockout mice.

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 alpha-calcitonin gene-related peptide (alpha-CGRP) gene. Although the beta-calcitonin gene-related peptide (beta-CGRP) gene is intact in these mice, alpha-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 alpha-CGRP on baseline BP and beta-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 alpha-CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, beta-CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that alpha-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.

Reciprocal role of the AT1 receptor in modulating renal and neuronal AT1 mRNA expression.

This study was designed to explore the mechanisms mediating the expression of the type 1 angiotensin II (AngII) receptor (AT1) in neuronal and renal tissues. Four groups of rats were given 1% NaCl in water and subjected to the renal reduced mass protocol (RRM), RRM + ramipril (Ram, 10 mg/kg per d), RRM + candesartan (Can, 10 mg/kg per d), or sham surgery. After 12 d, mean arterial pressure (MAP) was significantly higher in RRM rats than in RRM + Ram, RRM + Can, and sham-operated rats. Northern blot analysis showed that renal AT1 receptor mRNA levels (AT1 mRNA/18 rRNA) were significantly decreased in RRM (1.08+/-0.05) and RRM + Ram (0.82+/-0.02) compared with sham-operated rats (1.38+/-0.06) and that candesartan treatment caused a further decrease in renal AT1 mRNA content (0.73+/-0.07) compared with RRM. In contrast, dorsal root ganglia AT1 receptor mRNA content was significantly decreased in RRM (0.52+/-0.06) compared with sham-operated rats (1.18+/-0.07), and this decrease was abolished by ramipril (1.40+/-0.13) and candesartan treatment (1.56+/-0.11). RIA showed that levels (ng/mg protein) of calcitonin gene-related peptide (CGRP) in the dorsal root ganglia were significantly increased in RRM (1.60+/-0.11) but not in RRM + Ram (1.14+/-0.20) and RRM + Can (1.18+/-0.09), compared with sham-operated rats (0.94+/-0.05). Thus, RRM-induced downregulation of neuronal AT1 mRNA expression is mediated by AngII activation of the AT1 receptor, whereas an AT1-independent mechanism is operant in mediating renal AT1 gene expression. Furthermore, the inverse relationship between neuronal AT1 expression and CGRP content indicates that activation of the neuronal AT1 receptor inhibits CGRP synthesis in the dorsal root ganglia. The functional implications of these findings are discussed.

Pregnancy and steroid hormones enhance the vasodilation responses to CGRP in rats.

We recently reported that calcitonin gene-related peptide (CGRP) reversed the hypertension induced by nitric oxide inhibition in pregnant rats and that this effect appeared to be progesterone dependent. In the present study, we examined whether the vasodilator responses to CGRP are increased during pregnancy and whether these responses are steroid hormone dependent. Three groups of ovariectomized (Ovx) rats (n = 4-8 rats/group) were studied 3 days after daily treatment (subcutaneous injection) with progesterone (P; 2 mg/injection, twice daily for 3 days, in 0.2 ml of sesame oil), 17beta-estradiol (E; 2.5 microgram/injection, twice daily for 3 days, in 0.2 ml of sesame oil), or vehicle (sesame oil). A fourth group (n = 6 rats) of pregnant rats was studied on day 19 of gestation. A fifth group of adult, nonpregnant rats (n = 6 rats), regardless of stage of estrous cycle, was also used in this study. Mean arterial blood pressure (MAP) was continuously monitored in fully awake and free-moving instrumented rats. MAP was measured before and after administration of either saline or varying bolus doses of CGRP (9-360 pmol/kg body wt). CGRP produced a dose-dependent decrease in MAP in all rats with a significant (P < 0.05) reduction in MAP beginning with a CGRP dose of 90 pmol/kg and with maximal effects observed at 360 pmol/kg. Decreases in MAP in response to CGRP were significantly (P < 0.05) greater in pregnant compared with nonpregnant rats. Similarly to pregnant rats, Ovx rats given both E and P treatments produced greater decreases in MAP in response to CGRP at 90, 180, and 360 pmol/kg doses compared with both ovary-intact and Ovx nonpregnant rats, which were not different from each other. In summary, these data show that 1) the hypotensive effects of CGRP are dose dependent and 2) the hypotensive effects of CGRP are enhanced during pregnancy and in Ovx rats treated with either E or P. Therefore, we suggest that the decrease in vascular tone that is seen during pregnancy may be mediated, at least in part, by a sex steroid hormone-induced increase in the vascular sensitivity to the vasodilator effects of CGRP.

Adrenal medulla.

Although approximately 90% of patients diagnosed with hypertension have essential hypertension, it is important that secondary hypertension is considered; diagnosed, when present; and managed either medically or surgically. Although the most common organ involved in the etiology of secondary hypertension is the kidney, disorders of the adrenal gland also play a major role. This review focuses on recent findings and controversies about the adrenal medulla and, where appropriate, on the role of the adrenal medulla in hypertension.

Alpha 2-adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons.

Calcitonin gene-related peptide (CGRP), a potent vasodilator, is produced in dorsal root ganglia (DRG) neurons which extend nerves peripherally to blood vessels and centrally to the spinal cord. We previously reported that neuronal CGRP expression is significantly reduced in the spontaneously hypertensive rat (SHR) which could contribute to the elevated BP. Other studies suggest that the enhanced activity of the sympathetic nervous system in the SHR may mediate, at least in part, this reduction in neuronal CGRP expression via activation of alpha 2-adrenoreceptors (alpha 2-AR) on DRG neurons. To test this hypothesis in vitro we employed primary cultures of adult rat DRG neurons. Neuronal cultures were initially exposed (24 h) to either the alpha 2-AR agonist UK 14,304 (10(-6) M) or vehicle; however, no changes in CGRP mRNA content or immunoreactive CGRP (iCGRP) release were observed. Using the rationale that in vivo DRG neurons receive a continuous supply of target tissue derived nerve growth factor (NGF), which stimulates CGRP synthesis, the cultured neurons were treated (24 h) with either vehicle, NGF (25 ng/ml) alone, or NGF plus UK. NGF treatment increased CGRP mRNA accumulation 5.5 +/- 0.9-fold (p < 0.001) and iCGRP release 2.9 +/- 0.4-fold (p < 0.001) over control levels. The stimulatory effects of NGF were markedly attenuated, but not abolished, by UK (NGF + UK vs. control, CGRP mRNA, 2.9 +/- 0.4-fold, p < 0.05; iCGRP, 1.7 +/- 0.2-fold, p < 0.05). These values were also significant (p < 0.05) when compared to NGF treatment alone. Experiments performed using the alpha 2-antagonist yohimbine confirmed that the effects of UK were mediated by the alpha 2-AR. These results, therefore, demonstrate that alpha 2-AR activation attenuates the stimulatory effects of NGF on CGRP expression in DRG neurons.

Sugar-induced blood pressure elevations over the lifespan of three substrains of Wistar rats.

OBJECTIVE: Since the majority of studies concerned with sugar-induced blood pressure elevation have principally been short-term, the present investigation followed the effects of heavy sucrose ingestion on systolic blood pressure (SBP) and related parameters over the lifespan of three substrains of Wistar rats. METHODS: Two hundred twenty-five rats (75 spontaneously hypertensive rats (SHR), 75 Wistar Kyoto rats (WKY), 75 Munich Wistar rats (WAM) were given one of five diets. The baseline diet in terms of calories derived 32% from sucrose, 33% from protein, and 35% from fat. The remaining four diets derived their calories as follows: a high sugar-low protein diet--52% of calories from sucrose, 15% from protein, and 33% from fat; a high sugar-low fat diet--53% of calories from sucrose, 37% from protein, and 10% from fat; a low sugar-high protein diet--11% calories from sucrose, 56% from protein, and 33% from fat, and a low sugar-high fat--13% of calories from sucrose, 32% from protein, and 55% from fat. RESULTS: All substrains showed the highest systolic blood pressure when ingesting the two diets highest in sucrose. The highest sugar-induced SBP elevation, which remained over the lifespan of all substrains, was found in SHR. WKY had an intermediate elevation. WAM showed the lowest responses, although the average elevation of 6-8 mm Hg was statistically significant. The following parameters could not be correlated with long-term elevation of SBP; body weight, catecholamine excretion, renal function, and plasma renin activity. Only insulin concentrations correlated: insulin concentrations were consistently higher in the two groups of WKY and WAM consuming the high sucrose diets. CONCLUSIONS: High dietary sucrose can chronically increase SBP in three substrains of Wistar rats. Increased concentrations of circulating insulin were found in WKY and WAM suggesting that the glucose/insulin system was involved, at least in these two substrains, in the maintenance of high SBP levels during chronic, heavy sugar ingestion.

Calcitonin gene-related peptide is a depressor in subtotal nephrectomy hypertension.

We previously demonstrated that the neuronal expression of calcitonin gene-related peptide (CGRP), a potent vasodilator, is increased in deoxycorticosterone-salt-induced hypertension where it acts as a compensatory vasodilator to attenuate the elevated blood pressure. To determine whether CGRP is playing a similar role in subtotal nephrectomy-salt-induced hypertension, hypertension was induced in Sprague-Dawley rats (n=6) by subtotal nephrectomy and 1.0% saline drinking water. Control rats (n=6) were sham operated and given tap water to drink. CGRP(8-37), a CGRP receptor antagonist, was used to assess the hemodynamic role of CGRP in this setting. CGRP mRNA and peptide levels in dorsal root ganglia were also determined. Three weeks after either 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. CGRP(8-37) (3.2 or 6.4 x 10(4) pmol/L in 0.1 mL saline) and vehicle were administered intravenously to all rats. Baseline mean arterial pressure was higher in the subtotal nephrectomized rats compared with the controls (173+/-5 versus 113+/-5 mm Hg, P<.001). Vehicle administration did not change mean arterial pressure in either group, and CGRP(8-37) administration did not alter mean arterial pressure in the normotensive group. In contrast, CGRP(8-37) administration to the subtotal nephrectomized rats rapidly increased the already elevated mean arterial pressure at both the 3.2 x 10(4) pmol/L dose (7.8+/-1.1 mm Hg, P<.05) and the 6.4 x 10(4) pmol/L dose (9.6+/-0.8 mm Hg, P<.01). CGRP mRNA and peptide levels in the dorsal root ganglia were not significantly different between the two groups. These data suggest that in subtotal nephrectomy-salt-induced hypertension, CGRP may play a compensatory depressor role in an attempt to lower the elevated blood pressure.

Creating and evaluating an independent ambulatory internal medicine clerkship.

Patient care is shifting from an inpatient setting to an ambulatory setting. Despite this shift, most internal medicine clerkships provide the majority of medical student training in inpatient settings or in university tertiary care clinics, which are not representative of patient care in a community setting. We created a separate ambulatory clerkship that used volunteer community faculty at local and distant sites. The steps involved are described here, including finding time within the clerkship, reaching consensus within the department, defining the curriculum, identifying sites, and developing preceptors. Various parameters were measured to ensure quality in educational design. Comparisons of the 1-year pilot program, the full implementation program, and the inpatient program revealed that use of community sites does not affect cognitive knowledge acquisition but does influence students' satisfaction level.