Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance.

The present study determines if (1) basal protein levels of nitric oxide (NO) synthases (eNOS, iNOS, and nNOS) are different in cisplatin-sensitive (OV2008) and counterpart cisplatin-resistant (C13(*)) human ovarian cancer cells, (2) cisplatin alters NOS levels, (3) NO donor causes apoptosis and p53 upregulation, (4) NO donor sensitizes C13(*) cells to cisplatin via p53 upregulation (determined by p53 siRNA gene-knockdown), and (5) inhibition of endogenous NOS alters cisplatin-induced apoptosis. Basal iNOS levels were higher in OV2008 cells than in C13(*) cells. Cisplatin upregulated iNOS, but dramatically reduced eNOS and nNOS, in OV2008 cells only. Failure of cisplatin to upregulate iNOS and downregulate eNOS/nNOS in cisplatin-resistant C13(*) cells may be an aetiological factor in the development of resistance. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased p53 protein levels and induced apoptosis in both cell types, and enhanced cisplatin-induced apoptosis in C13(*) cells in a p53-dependent manner (i.e., enhancement blocked by p53 siRNA). Specific iNOS inhibitor 1400W partially blocked cisplatin-induced apoptosis in OV2008 cells. In cisplatin-resistant C13(*) cells, blocking all NOSs with N(G)-amino-L-arginine dramatically changed these cells from cisplatin-resistant to cisplatin-sensitive, greatly potentiating cisplatin-induced apoptosis. The data suggest important roles for the three NOSs in regulating chemoresistance to cisplatin in ovarian cancer cells.

Regulation of p53 and suppression of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian cancer cells.

Dysregulated apoptosis plays a critical role in the development of a number of aberrant cellular processes, including tumorigenesis and chemoresistance. However, the mechanisms that govern the normal apoptotic program are not completely understood. Soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP) promote mammalian cell viability via an unknown mechanism and p53 status is a key determinant of cell fate in human ovarian cancer cells. Whether an interaction exists between these two determinants of cell fate is unknown. We hypothesized that basal sGC activity reduces p53 content and attenuates p53-dependent apoptosis in human ovarian cancer cells. Suppression of sGC activity with the specific inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) lowered cGMP content, and increased p53 protein content and induced apoptosis in three ovarian cancer cell lines, effects which were attenuated by the cGMP analog 8-Br-cGMP and by Atrial Natriuretic Factor, an activator of particulate guanylyl cyclase, which circumvent the inhibition of sGC. ODQ prolonged p53 half-life, induced phosphorylation of p53 on Ser15, and upregulated the p53-dependent gene products p21, murine double minute-2, and the proapoptotic, p53-responsive gene product Bax. ODQ activated caspase-3, and ODQ-induced apoptosis was inhibited by overexpression of X-linked inhibitor of apoptosis Protein. Pretreatment with the specific p53 inhibitor pifithrin or downregulation of p53 using a specific small inhibitory RNA significantly attenuated ODQ-induced apoptosis. Moreover, ODQ-induced upregulation of p21 and Bax and ODQ-induced apoptosis were significantly reduced in a p53 mutant cell line relative to the wild-type parental cell line. Thus, the current study establishes that basal sGC/cGMP activity regulates p53 protein stability, content, and function, possibly by altering p53 phosphorylation and stabilization, and promotes cell survival in part through regulation of caspase-3 and p53.

Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta.

Synthetic atriopeptin II, an atrial natriuretic factor with potent vasodilatory effects, was studied in isolated strips of rat thoracic aorta to determine its actions on contractility, cyclic nucleotide concentrations and endogenous activity of cyclic nucleotide-dependent protein kinases. Atriopeptin II was found to relax aortic strips precontracted with 0.3 microM norepinephrine whether or not the endothelial layer was present. Relaxation to atriopeptin II was closely correlated in a time- and concentration-dependent manner with increases in cyclic GMP concentrations and activation of cyclic GMP-dependent protein kinase (cyclic GMP-kinase). The threshold concentration for all three effects was 1 nM. Atriopeptin II (10 nM for 10 min) produced an 80% relaxation, an 8-fold increase in cyclic GMP concentrations and a 2-fold increase in cyclic GMP-kinase activity ratios. Atriopeptin II did not significantly alter cyclic AMP concentrations or cyclic AMP-dependent protein kinase activity. These data suggest that cyclic GMP and cyclic GMP-kinase may mediate vascular relaxation to a new class of vasoactive agents, the atrial natriuretic factors. Similar effects have been observed with the nitrovasodilator, sodium nitroprusside, and the endothelium-dependent vasodilator, acetylcholine. Therefore, a common biochemical mechanism of action that includes cyclic GMP accumulation and activation of cyclic GMP-kinase may be involved in vascular relaxation to nitrovasodilators, endothelium-dependent vasodilators and atrial natriuretic factors.

Effects of atriopeptin on particulate guanylate cyclase from rat adrenal.

Atriopeptin II activated particulate guanylate cyclase 5-10-fold in a concentration- and time-dependent fashion in crude membranes obtained from homogenates of rat adrenal cortex or medulla. Similar effects were observed with other atriopeptin analogs. Soluble guanylate cyclase and adenylate cyclase in these preparations were not activated. Accumulation of cyclic GMP in minces of adrenal cortex or medulla was increased 6-8-fold due to atriopeptin II activation of particulate guanylate cyclase. Several thiol-reactive agents blocked the activation of particulate guanylate cyclase, suggesting that free thiol groups on membrane proteins may be important in atriopeptin receptor-guanylate cyclase coupling.

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside.

Methacholine (3 microM) and sodium nitroprusside (300 microM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 microM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0 degree C) and with an abbreviated incubation time (2.5 min). When assayed at 30 degrees C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 microM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0 degree C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.

Regulation of particulate guanylate cyclase by atriopeptins: relation between peptide structure, receptor binding, and enzyme kinetics.

Structural analogs of atriopeptins (APs) were compared for their ability to activate particulate guanylate cyclase and bind to specific receptors in rat adrenal membranes. All analogs tested increase Vmax without altering the concentration of substrate required for half-maximum activity or the positive coperativity exhibited by the enzyme. Maximum velocities (pmoles of cGMP produced per min per mg protein) achieved in the absence and presence of APs were 128.3 +/- 6.6 and 283.8 +/- 20.6 using Mn2+-GTP, and 53.7 +/- 3.7 and 149.9 +/- 7.6 using Mg2+-GTP as the substrate, respectively. Although all APs were equally efficacious in activating the enzyme, their rank potency was ANF (8-33) = AP III = AP II greater than AP I when either divalent cation was used as the cofactor. The EC50 for activation of guanylate cyclase by AP I was about 10(-7) M, while that for the other peptides was about 10(-8) M, using either divalent cation cofactor. 125I-labeled ANF bound to rat adrenal membranes with a KD of 5.10(-10) M. Although all APs were equally efficacious in competing with labeled ANF for receptor binding, their rank potency was identical to that for enzyme activation. The Ki for AP I was about 10(-8) M, while that for the other peptides was about 10(-10) M. These data suggest that the carboxy terminal Phe-Arg present in the AP analogs except AP I and critical for biological and receptor-binding activity are also important in coupling receptor-ligand interaction with guanylate cyclase activation. The correlation between the rank order potency for receptor binding, enzyme activation, and the reported physiological actions of APs support the suggestion of a functional coupling between these proteins.

Rapid nitric oxide- and prostaglandin-dependent release of calcitonin gene-related peptide (CGRP) triggered by endotoxin in rat mesenteric arterial bed.

1. Our objective was to determine whether endotoxin (ETX) could directly trigger the release of calcitonin gene-related peptide (CGRP) from perivascular sensory nerves in the isolated mesenteric arterial bed (MAB) of the rat and to determine whether nitric oxide (NO) and prostaglandins (PGs) are involved. 2. ETX caused time- and concentration-dependent release of CGRP, and as much as a 17 fold increase in CGRP levels in the perfusate at 10-15 min after the administration of ETX (50 micrograms ml-1). 3. CGRP-like immunoreactivity in the perfusate was shown to co-elute with synthetic rat CGRP by reverse-phase h.p.l.c. 4. Pretreatment of MAB with capsaicin or ruthenium red inhibited ETX-induced CGRP release by 90% and 71%, respectively. ETX-evoked CGRP release was decreased by 84% during Ca2(+)-free perfusion. 5. The release of CGRP evoked by ETX was enhanced by L-arginine by 43% and inhibited by N omega-nitro-L-arginine (L-NOARG) and methylene blue by 37% and 38%, respectively. L-Arginine reversed the effect of L-NOARG. 6. Indomethacin and ibuprofen also inhibited the ETX-induced CGRP release by 34% and 44%, respectively. No additive inhibition could be found when L-NOARG and indomethacin were concomitantly incubated. 7. The data suggest that ETX triggers the release of CGRP from capsaicin-sensitive sensory nerves innervating blood vessels. The ETX-induced CGRP release is dependent on extracellular Ca2+ influx and involves a ruthenium red-sensitive mechanism. Both NO and PGs appear to be involved in the ETX-induced release of CGRP in the rat mesenteric arterial bed.

Increase of calcitonin gene-related peptide (CGRP) release and mRNA levels in endotoxic rats.

Previously we showed that calcitonin gene-related peptide (CGRP) is released into the circulation during pathogenesis of septic shock in humans and endotoxicosis in rats and pigs. The present study examines the changes of both CGRP release and synthesis during endotoxicosis in rats. Endotoxin was administered as a bolus (5 mg/kg,i.v.) to rats lightly anesthetized with ether. Blood samples and lumbar dorsal root ganglia (DRG), neuronal cell bodies synthesizing CGRP, were taken from rats at 0, .5, 3, or 8 h after endotoxin administration. CGRP levels in plasma and the steady-state levels of mRNA for CGRP in DRG were determined by methods of radioimmunoassay and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), respectively. Endotoxin elevated plasma CGRP by 62%, but not mRNA at .5 h. Endotoxin increased plasma CGRP by 142% and CGRP mRNA in DRG by 36% at 3 h, and further increased plasma CGRP by 216% and CGRP mRNA in DRG by 88% at 8 h. The data suggest that both CGRP expression and release in sensory nerves are increased during development of endotoxicosis in rats. CGRP synaptic transmission may be maintained during the course of endotoxicosis in rats. DRG undergo an elevation of CGRP expression in response to endotoxicosis, presumably as an adaptation of CGRP release during the inflammation. CGRP, because of its extremely high potency in hypotensive, tachycardiac and immunosuppressive effects, may play an important role in the pathogenesis of septic shock.

Early endotoxic shock results in enhanced vasodilator responses to nitroglycerin but unaltered responses to neuropeptides calcitonin gene-related peptide and substance P.

To determine the role that vasoactive neuropeptides, calcitonin gene-related peptide, and substance P play in tissue-blood flow regulation during early septic shock, we examined the responsiveness of arteries removed from pigs 3 h after administration of Escherichia coli lipopolysaccharide or saline vehicle. The carotid, cranial mesenteric, and left anterior descending coronary arteries were excised, and rings were cut from each vessel. Constrictor responses were obtained to cumulative doses of norepinephrine or potassium chloride. Rings were reconstricted and challenged with acetylcholine, substance P, calcitonin gene-related peptide, and nitroglycerin. Lipopolysaccharide significantly increased the cranial mesenteric artery's response to high concentrations of norepinephrine and the response to nitroglycerin in all vessels. This enhancement of responses to nitroglycerin suggests augmented smooth-muscle responsiveness to an exogenous source of nitric oxide, possibly associated with early depression of basal endothelial function. Depression of agonist-induced nitric oxide release may mask such enhancement with endothelial-dependent dilators and may enhance the response to adrenergic constrictors in some vascular beds.

Calcitonin gene-related peptide release in endotoxicosis may be mediated by prostaglandins.

Three cyclo-oxygenase inhibitors (ibuprofen, indomethacin, and high dose aspirin) and two inhibitors of thromboxane biosynthesis (imidazole and low dose aspirin) were used to evaluate the role of prostaglandins and thromboxane in the release of calcitonin gene-related peptide (CGRP) during endotoxicosis. Endotoxin (lipopolysaccharide B from Salmonella Enteritidis, 5 mg/kg, intravenously) was administered to rats lightly anesthetized with ether during injection. After 3 h, endotoxin significantly elevated plasma CGRP levels by 3-fold. Ibuprofen (50 mg/kg, subcutaneously), indomethacin (10 mg/kg, subcutaneously) and high dose aspirin (100 mg/kg, intraperitoneally (i.p.)), but not imidazole (30 mg/kg, i.p.) or low dose aspirin (15 mg/kg, i.p.), significantly blocked endotoxin-induced CGRP elevations, suggesting that a prostaglandin, but not thromboxane, served as a mediator of CGRP release during endotoxicosis. Because endotoxin-induced production of prostaglandins is greatly diminished in endotoxin-tolerant rats (following multiple exposures to low dose endotoxin), we tested whether endotoxin-induced CGRP release also becomes diminished in tolerant rats. Accumulation of plasma CGRP was greatly diminished in endotoxin-tolerant rats exposed to endotoxin (5 mg/kg, intravenously), consistent with a mediator role for prostaglandins in the CGRP release during endotoxicosis.

Regional concentrations of cyclic nucleotides after experimental brain injury.

Regional concentrations of lactate, glucose, cAMP, and cGMP were measured after lateral fluid percussion brain injury in rats. At 5 min after injury, while tissue concentrations of lactate were elevated in the cortices and hippocampi of both the ipsilateral and contralateral hemispheres, those of glucose were decreased in these brain regions. By 20 min after injury, increases of lactate concentrations and decreases of glucose concentrations were observed only in the cortices and in the hippocampus of the ipsilateral hemisphere. Whereas the cAMP concentrations were unchanged in the cortices and hippocampi of the ipsilateral and contralateral hemispheres at 5 min after injury, decreases were found in the injured cortex and ipsilateral hippocampus at 20 min after injury. The tissue concentrations of cGMP were found to be elevated only in the ipsilateral hippocampus at 5 min after injury. The present observation that tissue glucose decreases in the injured cortex and the ipsilateral hippocampus are consistent with the published findings of increased hyperglycolysis and oxidative metabolism in brain immediately after injury. The present findings that the concentrations of cAMP and cGMP change in the cortex and hippocampus provide biochemical evidence for the neurotransmitter's surge after brain injury.

Plasma calcitonin gene-related peptide and atrial natriuretic peptide levels during resection of pheochromocytoma.

Calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) are potent hypotensive agents. To determine if they play a counterregulatory role in catecholamine excess in patients with pheochromocytoma, plasma levels were measured in four patients undergoing resection of sporadically occurring tumors. Each patient was prepared with phenoxybenzamine hydrochloride (Dibenzyline); two patients also received propranolol. Blood was obtained for plasma levels of epinephrine, norepinephrine, CGRP, and ANP at induction of anesthesia, skin incision, tumor manipulation, tumor removal, and 24 hours after operation. Baseline plasma norepinephrine and epinephrine levels were markedly elevated and increased significantly with tumor manipulation and decreased significantly 24 hours after operation. CGRP and ANP levels were slightly elevated throughout but did not change significantly with tumor manipulation or early after tumor resection. Circulating CGRP and ANP do not appear to have an acute counterregulatory role in catecholamine excess in patients with pheochromocytoma but may exert some influence on postoperative hypotension after tumor removal.

Calcitonin gene-related peptide levels are elevated in patients with sepsis.

Calcitonin gene-related peptide (CGRP), an endogenous vasoactive peptide encoded by the calcitonin gene in nerve cells, is distributed throughout the cardiovascular system and is a potent vasodilator. Plasma levels of CGRP have been elevated in animal models with sepsis. This study was designed to determine whether plasma CGRP levels are elevated in patients with sepsis and perhaps contribute to the hyperdynamic cardiovascular state in sepsis. Plasma CGRP levels were obtained from normal healthy volunteers and from patients with sepsis. Volunteers were afebrile and had normal pulse and blood pressure. Patients with sepsis were selected according to the following criteria: (1) temperature higher than 38.5 degrees C, (2) white blood count greater than 14,000/ml, (3) positive blood culture of bacterial organisms, (4) hemodynamic parameters consistent with hyperdynamic sepsis, and (5) negative history of thyroid or other endocrine abnormalities. CGRP was extracted and assayed by radioimmunoassay for iodine 125-labeled human CGRP. In patients with sepsis, the cardiac index was 5.4 +/- 0.5 L/min/m2 (normal, 3.0); systemic vascular resistance was 7.1 +/- 0.5 mm Hg/L/min (normal, 16); oxygen delivery was 1496 +/- 137 ml/min (normal, 1000). Plasma CGRP levels were significantly elevated in the patients with sepsis, 14.9 +/- 3.2 pg/ml, compared to plasma CGRP levels in control volunteers, 2.0 +/- 0.3 pg/ml (p less than 0.0005). These elevated levels of CGRP may contribute to the decreased vascular resistance and increased cardiac output in the hyperdynamic septic state.

Natriuretic peptides inhibit apoptosis and prolong the survival of serum-deprived PC12 cells.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were investigated to determine effects on apoptotic DNA fragmentation and survival in serum-deprived PC12 cells. Both peptides caused prolonged cGMP (but not cAMP) elevations lasting for > or = 6 h. The cGMP elevations were 10-, 50- and 68-fold for ANP and 26-, 100- and 148-fold for BNP at 1, 10 and 100 nM, respectively. BNP caused dose-dependent increases in cell survival rates during 3 days of serum deprivation. BNP (1 nM) increased 24 h survival rate from 36% to 67%. ANP (1 nM), BNP (1 nM) and 8-bromo-cGMP (0.1 mM) inhibited by 74.8%, 46.7% and 86.8%, respectively, the apoptotic DNA fragmentation in serum-deprived PC12 cells, measured by our recently developed quantitative technique using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF). The data suggest prolonged cGMP elevations caused by ANP or BNP inhibit apoptotic DNA fragmentation and prolong the survival of serum-deprived PC12 cells.

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats.

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.

RGD-containing peptides induce endothelium-dependent and independent vasorelaxations of rat aortic rings.

Peptides containing the extracellular matrix peptide cell attachment sequence RGD possess potent, endothelium-dependent vasorelaxant properties. In the present study, the ability of RGD-containing peptides to cause vasorelaxation in the presence and absence of a functional endothelium was examined in rat aortic rings along with the ability of RGD-containing peptides to increase cGMP production in these vessels. The active RGD-containing peptide GRGDNP induced rapid relaxation in endothelium-intact, norepinephrine contracted rat aortic rings. When the endothelium was removed, RGD-containing peptides produced a slow relaxation of contracted rings which took approx. 40 min to reach maximum relaxation. Control RGD peptides were without effect either in the presence or absence of a functional endothelium. While acetylcholine and sodium nitroprusside stimulated cGMP production in endothelium-intact and denuded aortic segments, neither the control RGD peptide nor the active GRGDNP increased cGMP in these vessels when compared to controls upon either short (30 s) or long (45 min) incubation times. These data indicate that relaxations of rat aortic rings in response to RGD-containing peptides occur both in the presence and absence of an intact endothelium and that cGMP is likely not the sole mediator of these responses.

Brain natriuretic peptide (BNP) causes endothelium-independent relaxation and elevation of cyclic GMP in rat thoracic aorta.

The novel neuropeptide, brain natriuretic peptide (BNP), causes concentration-dependent relaxations in rat isolated arterial rings. The pD2 value of BNP in rat thoracic aorta is 8.05 +/- 0.06, almost identical to the pD2 value of atrial natriuretic peptide (the 28 amino acid peptide, rat sequence, AP-28, 8.11 +/- 0.08), indicating that BNP and ANP have the same potency in relaxing thoracic aorta. In addition, BNP is equally potent at causing relaxation in abdominal aorta and mesenteric and renal arteries. However, BNP is less potent in causing vasorelaxation in the common iliac and femoral arteries and shows no relaxant effects in caudal arteries. This pharmacological profile of BNP in different rat arteries is very similar to that of ANP. Like ANP, BNP induces a vasorelaxation that is independent of endothelium and is associated with very sustained increases in cyclic GMP, but not cyclic AMP, levels in rat thoracic aorta. The BNP-induced cyclic GMP elevation, like the vasorelaxation, is also independent of endothelium and is not blocked by methylene blue (10 microM), a soluble guanylate cyclase inhibitor. Furthermore, BNP-induced cyclic GMP elevation is independent of extracellular calcium and potentiated by the cyclic GMP-phosphodiesterase inhibitor M & B 22948. Therefore, the pharmacological characteristics of BNP in rat blood vessels are very similar to those of ANP, suggesting that BNP and ANP may act through a common receptor and post-receptor mechanism to cause vasodilation.

Calcitonin gene-related peptide causes long-term inhibition of contraction in rat thoracic aorta through a nitric oxide-dependent pathway.

Calcitonin gene-related peptide (CGRP) is released into the circulation during pathogenesis of endotoxin and septic shock and appears to partly mediate vascular problems of shock. To explore the function of CGRP during shock, we investigated long-term action of CGRP, alone or in combination with interleukin-1beta (IL-1beta), another shock mediator, in isolated rings of rat thoracic aorta. CGRP or IL-1beta, by themselves, caused significant long-term (3 h) depression of contraction, while the combination of CGRP and IL-1beta had no synergistic effects. Dose-response curves to phenylephrine were significantly decreased and shifted to the right when aortic rings were incubated with 1 microM CGRP for 1 h followed by 2 h incubation without CGRP. Inducible nitric oxide synthase (iNOS) inhibitors, S-methylisothiourea sulfate (SMT) and N(G)-nitro-L-arginine (L-NNA), completely eliminated long-term depressant effect of CGRP. Our results suggest pathology of septic shock may involve long-term inhibition of vascular contraction mediated by CGRP via expression of iNOS.

Calcitonin gene-related peptide (CGRP) causes endothelium-dependent cyclic AMP, cyclic GMP and vasorelaxant responses in rat abdominal aorta.

Calcitonin gene-related peptide (CGRP), a neuropeptide found in nerves surrounding most blood vessels, is a potent hypotensive agent in both humans and rats. In isolated strips of rat thoracic aorta, CGRP has been reported to cause endothelium-dependent relaxation. To study the cellular and molecular mechanisms involved in CGRP-induced vasodilation, we investigated the roles of two second messengers, cyclic AMP and cyclic GMP, as potential mediators of the signal transduction mechanism leading to vasodilation in response to CGRP in rat aorta. In the present study, the abdominal aorta, rather than thoracic aorta, was used because of its higher content of endogenous CGRP and, therefore, the greater likelihood of regulation by CGRP in vivo. Each abdominal aortic ring was precontracted with norepinephrine (NE) at its EC50 concentration (10-20 nM). CGRP (3-300 nM) caused concentration-dependent relaxations (reducing the NE-induced contractions by 34%) that were completely dependent on endothelium. The relaxations in response to CGRP were correlated in a time- and concentration-dependent manner with increases in aortic levels of both cyclic AMP and cyclic GMP. CGRP (100 nM) caused significant elevations of cyclic AMP levels (1.4 to 3.2 pmol/mg protein, at 1 min) and cyclic GMP levels (1.6 to 3.6 pmol/mg protein, at 30 s). Like the vasorelaxant responses, both cyclic AMP and cyclic GMP responses to CGRP were totally dependent on the endothelium. Pre-incubation with indomethacin (3 microM, 15 min) did not alter cyclic AMP responses to CGRP (100 nM), suggesting that prostaglandins are not involved. Therefore, CGRP-induced vasodilations of abdominal aorta involve an endothelium-dependent mechanism associated with cyclic GMP elevations, similar to the mechanisms of vasodilation in response to acetylcholine and other endothelium-dependent vasodilators. However, CGRP-induced relaxations of aorta involve an additional mechanism (i.e., endothelium-dependent cyclic AMP elevations), which may also contribute to the intracellular mechanism of aortic vasodilation in response to CGRP.

N omega-nitro-L-arginine inhibits vasodilations and elevations of both cyclic AMP and cyclic GMP levels in rat aorta induced by calcitonin gene-related peptide (CGRP).

Our previous studies showed that vasodilations and elevations of both cyclic AMP and cyclic GMP levels in rat aorta induced by rat calcitonin gene-related peptide (rCGRP) are inhibited by hemoglobin and methylene blue, blockers of the endothelium-derived relaxant factor (EDRF, now recognized as nitric oxide [NO]). In the present study, we used N omega-nitro-L-arginine (L-NNA), a selective inhibitor of nitric oxide synthase, to test whether rCGRP-induced relaxations and cyclic AMP and cyclic GMP responses in rat aorta require de novo synthesis of NO. L-NNA (30 microM, 15 min) inhibited by 84, 76 and 73% the relaxations induced by rCGRP at 1, 10 and 100 nM, respectively. D-NNA (30 microM), which does not inhibit nitric oxide synthase, did not block rCGRP-induced vasorelaxations. Addition of L-arginine (3 mM) 5 min before L-NNA completely prevented the L-NNA-inhibition of CGRP-induced relaxations. L-NNA (30 microM, 15 min) also inhibited the elevations of both cyclic AMP and cyclic GMP levels caused by CGRP (100 nM). The data suggest that de novo synthesis of nitric oxide from its precursor L-arginine is required for rCGRP to induce vasodilations and elevations of both cyclic AMP and cyclic GMP levels in rat aorta.