Calcitonin gene-related peptide (CGRP)-induced cyclic AMP, cyclic GMP and vasorelaxant responses in rat thoracic aorta are antagonized by blockers of endothelium-derived relaxant factor (EDRF).

The mechanism of CGRP-induced vasodilation in rat thoracic aorta was investigated using antagonists of the classical endothelium-derived vasorelaxant factor (EDRF) and comparisons with acetylcholine-induced vasodilations. The CGRP-induced relaxations of isolated rings of rat thoracic aorta were completely dependent on the presence of endothelium and were associated with increases in the levels of both cyclic AMP and cyclic GMP, the same as in our previous study using rat abdominal aorta. Maximum relaxations to CGRP, which represented 40-50% reversal of the norepinephrine-induced contractions, occurred with 100 nM CGRP. Addition of acetylcholine (ACh, 1 microM) to aortic rings, which were already maximally relaxed to CGRP, caused further relaxation to 100%, suggesting that CGRP may use a mechanism (or pool of EDRF) different from that of ACh. Both CGRP- and ACh-induced relaxations of aorta were significantly inhibited by the EDRF blocking agents, hemoglobin (10 microM), methylene blue (10 microM), and nordihydroguaiaretic acid (NDGA, 10 microM). In fact, hemoglobin and NDGA were more effective as inhibitors of CGRP-induced relaxations than ACh-induced relaxations. Hemoglobin, methylene blue and NDGA also inhibited the CGRP-induced increases in both cyclic AMP and cyclic GMP levels. On the other hand, indomethacin, a cyclo-oxygenase inhibitor, did not alter CGRP-induced vasorelaxations or increases in either cyclic AMP or cyclic GMP levels, suggesting that prostaglandins are not involved. Therefore, CGRP-induced vasodilations in rat thoracic aorta appear to involve EDRF, leading to cyclic GMP elevations in smooth muscle and ultimately vasorelaxations. However, another previously undescribed mechanism, which involves EDRF-dependent and indomethacin-resistant elevations of cyclic AMP levels, is triggered by CGRP in thoracic aorta. This novel EDRF-dependent cyclic AMP response may contribute to the CGRP-induced vasodilation in rat thoracic aorta.     

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.     

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 induces the formation of second messengers in primary cultures of neonatal rat spinal cord

This study investigated second messengers formed in response to calcitonin gene-related peptide (CGRP) in primary cultures of neonatal rat spinal cord. CGRP increased the level of cAMP above basal levels (50 pmol/mg protein) over a large range of concentrations. The concentration-response curve had an intermediate plateau at 180 pmol cAMP/mg protein in response to 0.01–0.1 nM CGRP and a maximal plateau of 850 pmol cAMP/mg protein at 300 nM CGRP. The biphasic concentration-response curve (EC₅₀s of 0.7 pM and 22 nM) suggests activation of high- and low-affinity receptors for CGRP. Both neurons and nonneuronal cells contributed to the increase in cAMP formation in response to CGRP. The CGRP receptor blocker, CGRP_8–37, inhibited the response to both 1 and 100 nM CGRP, providing additional support for the hypothesis that both high- and low-affinity receptors mediate the formation of cAMP. Only a high concentration of CGRP (1 μM) increased the formation of cGMP, and CGRP had no effect on the formation of inositol phosphates at any of the concentrations tested (0.1–1 μM). These results suggest that CGRP-induced responses in the spinal cord are mediated predominately via the formation of cAMP. The observation that both neurons and nonneuronal cells responded to CGRP indicate that this peptide may have multiple actions in the spinal cord. Synapse 26:235–242, 1997. © 1997 Wiley-Liss Inc.     

Vasodilatory actions of calcitonin gene-related peptide and nitric oxide in parenchymal microvessels of the rat hippocampus

Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent dilators in a variety of vascular beds. Recent evidence suggests that NO may serve as an intermediary messenger for CGRP and/or CGRP may serve as an intermediary messenger for NO in the expression of vasodilation. The present study was designed to provide an initial characterization of the responses to NO and CGRP in parenchymal microvessels and to determine whether NO and/or CGRP act as intermediaries for one another. Microvessels in the parenchyma of in vitro hippocampal slices from rat brain were examined using computer-assisted videomicroscopy. The resting diameter of the microvessels ranged from 9 to 26 μm. Treatment with the nitric oxide synthase inhibitor, N^G-nitro-l-arginine ( -NNA; 100 μM) constricted vessels to 64.2% ± 3.0% of resting luminal diameter. Sodium nitroprusside (SNP; 1 μM), a donor of NO, reversed the -NNA-induced vasoconstriction by 77.0% ± 15.0%. CGRP alone (10 nM) elicited a small but significant vasodilatory effect on resting vascular tone (2.3% ± 0.6%). In the presence of -NNA, CGRP elicited a significant dose-dependent vasodilatory response, and 10 nM CGRP elicited a sizeable response, reversing the -NNA-induced constriction by 84.3% ± 15.5%. This CGRP-induced dilation was inhibited by pretreatment with the CGRP receptor antagonist, CGRP fragment (8–37) (1 μM). In contrast, pretreatment with 1 μM CGRP fragment (8–37) did not attenuate the SNP-induced dilation in the presence of -NNA. Taken together, these findings demonstrate that CGRP and NO are potent dilators of parenchymal microvessels, and that NO provides a substantial relaxant effect on resting tone. In addition, the results indicate that CGRP is not a necessary intermediary in NO-induced dilation, and that NO is not a necessary intermediary in CGRP-induced dilation in parenchymal microvessels.     

Inhibitory Effects of Natriuretic Peptides on Vasopressin Neurons Mediated through cGMP and cGMP-Dependent Protein Kinase in vitro

The effects of natriuretic peptides on electrical activity and cellular cGMP levels were studied in neurons of the supraoptic nucleus (SON) of rat hypothalamic slice preparations. Intracellular and extracellular recordings showed that bath application of A type natriuretic peptide (ANP) at 100 nM or B type natriuretic peptide (BNP) at 100 to 300 nM decreased the firing rate and hyperpolarized the membrane potential in phasically firing (putative vasopressin) neurons. Non-phasically firing (putative oxytocin) neurons did not respond to these natriuretic peptides in firing rate or membrane potential. The membrane-permeable cGMP analogue 8-bromo cGMP at 0.5 mM and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 50 μM mimicked the inhibitory effects of ANP and BNP. The specific inhibitor of cGMP phosphodiesterase 1-(3-chloroanilino)-4-phenylphthalazine (MY5445) at 30 μM also decreased the firing rate of SON neurons. The cGMP-dependent protein kinase inhibitor N-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide dihydrochloride (H8) at 1 μM abolished the inhibition by natriuretic peptides. We measured cGMP and cAMP contents in discrete SON regions and compared the change of the contents before and after application of ANP and BNP. The increases in cellular cGMP accumulation were 430% for ANP and 120% for BNP, although they did not cause significant change of cAMP accumulation. The results suggest that the inhibitory effects of natriuretic peptides on putative vasopressin neurons are mediated through cGMP and cGMP-dependent protein kinase.     

Neuropeptide effects on rat chondrocytes and perichondrial cells in vitro

This study examines if cultured chondrocytes and perichondrial cells change the level of cAMP and/or cGMP in response to application of the neuropeptide calcitonin gene-related peptide (CGRP). Cells collected from the knee region of 4-8 days old rat pups were cultured in vitro. Cultures were exposed to 10(-10)-10(-6) M CGRP during 10 minutes. The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the cultures and in controls were determined by radioimmunoassay. The results show that application of CGRP causes a distinctly increased level of cAMP, that was absent when CGRP was applied together with the CGRP(1) receptor antagonist. The level of cGMP was not obviously altered. Hence, it is possible that terminals of primary sensory neurones present in developing cartilage influence chondrocytes and perichondrial cells via local release of CGRP.     

Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma × glioma hybrid cells (NG108-15)

Adrenomedullin (ADM) is a hypotensive peptide with structural homology, including a ring structure linked by a disulfide bridge, to calcitonin gene-related peptide (CGRP), calcitonin and amylin. ADM is predominantly synthesized in the adrenal medulla, but immunoreactive ADM has also been detected in the human brain. Here we have characterized ADM binding sites in cultured rat astrocytes using human [¹²⁵I]ADM(1–52) as radioligand. Half-maximal inhibition of [¹²⁵I]ADM(1–52) binding by intact rat ADM(1–50) amounted to 0.27 ± 0.03 nM (n = 15). The related peptides rat α-CGRP, rat amylin and salmon calcitonin displaced [¹²⁵I]ADM(1–52) at 85-, 148-, and > 4000-fold higher concentrations. Half-maximal stimulation of CAMP accumulation by rat ADM(1–50) was obtained with 1.00 ± 0.12 nM (n = 16). Rat α-CGRP was 214-fold, and rat amylin and salmon calcitonin were > 1000-fold less potent. Concerning cAMP accumulation the results were indistinguishable in mouse neuroblastoma × rat glioma hybrid cells (NG108-15), but here rat α-CGRP was > 1000-fold less potent than rat ADM(1–50). Human ADM(22–52) and human CGRP-I(8–37), which lack the ring structure, failed to stimulate cAMP accumulation, but they antagonized rat ADM(1–50) stimulated cAMP accumulation with inhibitory constants of 365 ± 93 nM and 92 ± 2 nM in astrocytes, and 45 ± 3 nM and 1300 ± 500 nM in NG108-15 cells. Rat ADM(1–50) did not raise cytosolic free calcium concentrations in astrocytes and NG108-15 cells. In conclusion, we have identified novel ADM receptors coupled to cAMP formation in cultured rat astrocytes and NG108-15 cells. Different interactions with the homologous peptide CGRP as well as the truncated receptor antagonists ADM(22–52) and CGRP(8–37) in rat astrocytes and neuroblastoma × glioma hybrid cells are consistent with ADM receptor isotypes in the brain.     

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.     

Activators of cyclic adenosine 3':5'-monophosphate accumulation in rat hippocampal slices: action of vasoactive intestinal peptide (VIP)

The present experiments tested the ability of putative neurotransmitters and neuromodulators to regulate cyclic adenosine 3':5'-monophosphate (cAMP) levels in rat hippocampal slices. Slices from ovariectomized adult female rats were equilibrated for 1 hr and incubated for 20 min with various test compounds, and cAMP was extracted and quantified using a competitive protein-binding assay. Norepinephrine, adenosine, histamine, and prostaglandins E1 and E2 alpha, induced moderate (1.5- to 5-fold) increases in cellular cAMP, whereas dopamine, serotonin, prostaglandin F2 alpha, and glutamate were relatively ineffective. Most striking was the observation that vasoactive intestinal peptide (VIP) produced marked elevation (approximately 80-fold at 6 microM) of hippocampal slice cAMP content. In contrast, other peptides produced only 2-fold increased (glucagon, somatostatin) or no change in cellular cAMP levels (enkephalins, LHRH, ACTH analogue, arginine vasopressin). Significant elevations in cAMP were seen with VIP concentrations as low as 20 nM; the cAMP response was half-maximal at 1 microM VIP and maximized between 10 and 20 microM. At maximally effective concentrations, VIP was 86% as effective in increasing cAMP as maximal concentrations of forskolin, a compound which activates adenylate cyclase in most cell types. The cAMP response to 10 microM VIP was pronounced after a 1-min incubation (16-fold elevations) and was maximal at 30 min (140-fold elevation). When slices from other brain areas were compared, it was found that regions known to contain high levels of VIP (cerebral cortex) also responded to VIP treatment with 30- to 50-fold elevations in cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina

The effects of vasoactive intestinal peptide (VIP) on glutamate-induced delayed death were examined using the primary cultures of rat retinal neurons. Effects of VIP on glutamate-induced neurotoxicity were evaluated by double staining with fluorescein diacetate and propidium iodide. Glutamate (1 mM) was applied to the culture for 10 min in the presence and absence of VIP, and visible cells enumerated 24 h after culture in normal medium. Effects of VIP on increase in the intracellular Ca²⁺ concentration and currents induced by glutamate in retinal neurons were investigated using the Ca²⁺ image analyzing system with fura-2 and whole-cell patch-clamp recording, respectively. The cAMP contents in retinal cultures were measured by radioimmunoassay. VIP (10 nM–1 μM) dose-dependently protected against glutamate-induced neurotoxicity in cultured retinal neurons. Protection by VIP (100 nM) against glutamate (1 mM)-induced neurotoxicity was antagonized by VIP6-28 (1 μM), a VIP antagonist, and H-89 (100 nM and 1 μM), a protein kinase A inhibitor. However, VIP had no effect on glutamate-induced inward currents nor glutamate-induced increase in the intracellular Ca²⁺ concentration. A 10-min exposure of VIP (100 nM) with glutamate (1 mM) resulted in an increase in the cAMP level to 446±58 from 22±1 pmol/mg protein. These findings suggest that VIP protects against the glutamate-induced neurotoxicity in retinal cultures by elevating the cAMP level via VIP receptors and thereby activating protein kinase A.     

Modulation by protein kinase C of nitric oxide and cyclic GMP formation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells withl-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 ± 0.03 nmol/h/dish (2.5 × 10⁶ cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 ± 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with staurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg²⁺, N-methyl-d-aspartate and, to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C, an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.     

Glyceroltrinitrate facilitates stimulated CGRP release but not gene expression of CGRP or its receptor components in rat trigeminal ganglia

Nitric oxide (NO) donors induce delayed headaches in migraineurs. In a corresponding rat model NO donors cause delayed ongoing activity in central trigeminal neurons which process intracranial afferent input. Cellular models indicate that NO may increase the release or production of calcitonin gene-related peptide (CGRP), a key mediator in primary headaches. CGRP release from intact isolated trigeminal ganglia of adult male Wistar rats was investigated in vitro. Exposure to high NO donor concentrations did not affect basal or stimulated CGRP release. After a two hour infusion of the NO donor glyceroltrinitrate (250 μg/kg/h), however, inflammatory mediators-induced CGRP release was 80% higher compared to control animals. Administration of the soluble guanylate cyclase inhibitor ODQ or the application of 8Br–cGMP revealed a cGMP-independent mechanism. In four groups of separate experiments total mRNA was extracted from rat trigeminal ganglia up to 6 h after glyceroltrinitrate or saline infusion. Gene expression of CGRP and the CGRP-receptor components, receptor activity-modifying protein 1, receptor component protein and calcitonin receptor-like receptor was measured by quantitative RT-PCR. Glyceroltrinitrate infusion did not change mRNA levels of these genes compared to infusion of saline. The present data suggest that prolonged increase in NO levels facilitates stimulated CGRP release from trigeminal ganglion neurons. The underlying mechanism appears to be independent of the cGMP pathway and not to interact with CGRP in the trigeminal ganglion. Delayed headaches induced by NO may change CGRP or CGRP-receptor expression.     

Brain natriuretic peptide and fluid volume homeostasis—studies during cardiopulmonary bypass surgery

Brain natriuretic peptide (BNP) is a recently identified cardiac ventricular hormone with diuretic, natriuretic and vasorelaxant properties. The aim of our study was to examine whether serial changes in endogenous levels of BNP were associated with fluid volume homeostasis following cardiopulmonary bypass.We studied nine patients undergoing elective cardiac surgery for the repair of cardiac abnormalities requiring cardiopulmonary bypass. Urinary levels of cyclic guanosine monophosphate (cGMP), sodium, urine output, fluid balance, and plasma levels of BNP, aldosterone, plasma renin activity (PRA) and central venous pressure (CVP) were measured before, during and after cardiopulmonary bypass.Basal pre-operative plasma BNP levels were highly elevated in all nine patients with cardiac abnormalities. During bypass, pre-operative levels of BNP, urinary cGMP and plasma aldosterone decreased significantly (p < 0.05), whereas pre-operative levels of urinary sodium and PRA were slightly reduced. During recovery following bypass levels of urinary cGMP, sodium, PRA and aldosterone returned to basal pre-operative values, whereas post-operative levels of plasma BNP were found to be three-fold below basal pre-operative levels. CVP (4.3 ± 0.2 mmHg) during the onset of bypass increased significantly (p < 0.05) at the end of bypass (9 ± 0.3 mmHg) followed by a modest increase post-operatively (10 ± 0.4 mmHg). After operation only BNP had a significant correlation with urine output (r= –0.82,p < 0.02) and net fluid balance (r = –0.84,p < 0.01), whereas urinary cGMP, PRA and aldosterone all exhibited a non-significant correlation with urine output. The overall plasma BNP levels showed a non-significant positive correlation with urinary cGMP (r = 0.58,p = 0.12), CVP (r = 0.53,p = 0.3), aldosterone (r = 0.53,p = 0.2) and PRA (r = 0.63,p = 0.075). The strong association between changes in endogenous BNP with urine output and fluid balance provides evidence for involvement of BNP in the regulation of fluid volume homeostasis.     

Calcitonin gene-related peptide and cerebral blood vessels: distribution and vasomotor effects

The innervation of cerebral blood vessels by nerve fibers containing calcitonin gene-related peptide (CGRP) and the vasomotor effects of this peptide are described for a number of different mammalian species. CGRP-immunoreactive nerve fibers were present in the adventitia of cerebral arteries in all species examined (guinea pig, cat, rabbit, rat, and mouse). Numerous perikarya containing CGRP immunoreactivity are demonstrable in the trigeminal ganglion of all species. In the cerebral perivascular nerve fibers and in trigeminal perikarya, CGRP is often colocalized with substance P and neurokinin A. Marked interspecies differences exist both in the density of CGRP-immunoreactive nerve fibers and in the cerebrovascular levels measured with radioimmunoassay. The highest concentrations were observed in cerebral vessels from guinea pigs, the lowest concentration in rabbit vessels, and intermediate levels in the feline and human cerebral vasculature. CGRP is a potent dilator of cerebral arteries in all species examined (human pial, feline middle cerebral, rabbit, guinea pig and rat basilar arteries). The concentration of CGRP eliciting half-maximal responses ranged from 0.4 nM (human pial artery) to 3 nM (rat and rabbit basilar arteries). Pretreatment of cerebral arteries with low concentrations of either substance P (0.1 nM) or neurokinin A (3 nM) attenuated slightly the CGRP-induced relaxations of guinea pig basilar arteries. Calcitonin was found to be a very weak dilator of cerebral arteries from human and guinea pig. Thus, cardiovascular nerve fibers containing CGRP appear to be present in all mammalian species (although to varying degrees) and CGRP is invariably a potent dilator of the cerebral arteries for all species.     

Hypotension dilates pial arteries by KATP and Kca channel activation

Hypotension induced pial artery dilation is prostaglandin-dependent in the newborn pig. Prostaglandins, in turn, elicit vasodilation through cGMP and cAMP dependent mechanisms and K⁺ channel activation contributes to cyclic nucleotide induced vasodilation. The present study was designed to characterize the role of ATP sensitive (K_ATP) and calcium sensitive (K_ca) channel activation in hypotension induced pial artery dilation in newborn pigs equipped with a closed cranial window. Glibenclamide and iberiotoxin, K_ATP and K_ca channel antagonists, attenuated hypotension induced dilation (36±1 vs. 14±2% before and after iberiotoxin). Combined administration of these K⁺ channel antagonists eliminated the vascular response. Hypotension induced dilation was associated with elevated cerebrospinal fluid (CSF) cAMP but not cGMP concentration (1023±29 vs. 1566±39 fmol/ml for cAMP). L-NNA, a nitric oxide (NO) synthase inhibitor, and Rp 8-Br cGMPs, a protein kinase G inhibitor, had no effect but Rp 8-Br cAMPs, a protein kinase A inhibitor, attenuated hypotensive dilation (35±1 vs. 16±2% before and after Rp 8-Br cAMPs). Dilation by the cAMP analogue 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) was attenuated by glibenclamide and iberiotoxin (8±1 and 17±1 vs. 4±1 and 9±1% before and after glibenclamide). These data show that both K_ATP and K_ca channel activation contribute to hypotension induced dilation. These data suggest that dilation during hypotension results from the sequential release of prostaglandins and cAMP, which, in turn, activates both the K_ATP and K_ca channel.     

Contribution of Kca channel activation to hypoxic cerebrovasodilation does not involve NO

Although nitric oxide (NO) and calcium sensitive K⁺ channel (K_ca) activation contribute to hypoxic pial artery dilation in the piglet, responses to the NO releasers SNP and SNAP are unchanged by the K_ca channel antagonist iberiotoxin. These data suggest that NO does not elicit dilation via K_ca channel activation. The present study was designed to determine if dilation by K_ca channel activation is mediated by NO in newborn pigs equipped with a closed cranial window. NS1619 (10⁻⁸, 10⁻⁶ M), a K_ca agonist, produced dilation that was unchanged by the NO synthase inhibitor, -NNA (10⁻⁶ or 10⁻³ M) (11±1 and 20±1 vs. 11±1 and 18±1% before and after -NNA 10⁻³ M). NS1619 dilation also was not associated with increased CSF cGMP and was unchanged by Rp 8-Bromo cGMPs, a cGMP antagonist (9±1 and 17±1 vs. 9±1 and 16±2% before and after Rp 8-Bromo cGMPs 10⁻⁵ M). Iberiotoxin (10⁻⁷ M) attenuated hypoxic dilation but hypoxia associated CSF cGMP release was unchanged (418±11 and 897±31 vs. 419±10 and 896±25 fmol/ml for control and moderate hypoxia before and after iberiotoxin). Coadministration of -NNA with iberiotoxin further decremented hypoxic pial dilation and blocked the hypoxia-associated rise in CSF cGMP. These data show that pial artery dilation by K_ca channel activation is not mediated by NO/cGMP. Further, these data suggest that NO and the K_ca channel act at different sites in their contributions to hypoxic pial artery dilation.     

Adenosine increases human platelet levels of cGMP through nitric oxide: possible role in its antiaggregating effect

Adenosine is an endogenous antiaggregating substance that influences the platelet responses through specific A-type receptors that activate adenylate cyclase increasing the levels of 3',5'-cyclic adenosine monophosphate (cAMP). In this study, we investigated whether adenosine can also influence the levels of 3',5'-cyclic guanosine monophosphate (cGMP) and decrease the aggregating response of human platelets to adenosine-5-diphosphate (ADP) through this nucleotide. In platelet samples from healthy volunteers, we evaluated the effect of adenosine on ADP-induced aggregation and cyclic nucleotide synthesis. Some experiments were repeated in the presence of dipyridamole (inhibitor of adenosine uptake and phosphodiesterase activity), N(G)-monomethyl-L-arginine (L-NMMA, nitric synthase inhibitor), ionomycin (calcium ionophore), and ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine, inhibitor of nitric oxide (NO)-dependent activation of guanylate cyclase). Adenosine decreased the response to ADP in a concentration-dependent way (analysis of variance, ANOVA: P<.0001): cAMP levels increased from 30.0 +/- 2.0 (control) to 46.0 +/- 3.0 pmol/10(9) platelets (in the presence of 15 mumol/l adenosine) and cGMP levels increased from 5.6 +/- 1.0 (control) to 10.9 +/- 2.0 pmol/10(9) platelets (in the presence of 15 mumol/l adenosine). Also, nucleotide levels measured at the end of aggregation were higher in platelet samples exposed to adenosine than in controls. Dipyridamole at 40 mumol/l slightly increased adenosine's effects on both nucleotides. L-NMMA blunted the effect of adenosine on cGMP both in unstimulated samples and in aggregated platelets without any effect on cAMP synthesis. Platelet exposure to L-NMMA and ambroxol partially prevented adenosine's effect on ADP-induced aggregation. In conclusion, adenosine, which enhances intraplatelet cAMP levels, was determined to also cause an increase in cGMP concentrations through a mechanism that involves NO synthesis. This effect plays a direct role in the adenosine-induced antiaggregation.     

Natriuretic peptide receptor-A is functionally expressed on bullfrog retinal Müller cells

By the patch clamp technique, whole-cell currents induced by brain natriuretic peptide (BNP) from isolated bullfrog retinal Müller cells were studied. Application of 100 nM BNP induced a sustained inward current from these cells with a reversal potential of about 0 mV, and the current could be completely blocked by anantin, an antagonist of the A-type NP receptor (NPR-A) and CdCl(2), a blocker of cyclic nucleotide-gated (CNG) non-selective cation channels. Likewise, perfusion with the membrane-permeable cGMP analog 8-bromoguanosine-3',5'-cyclic monophosphate (8Br-cGMP) caused effects that are similar to those of BNP. Moreover, application of BNP failed to induce any current in the presence of 1 mM 8Br-cGMP. By calcium imaging, we further showed a significant increase in intracellular calcium levels ([Ca(2+)](i)) of all parts of Müller cells, including the endfoot, soma and processes following the perfusion of BNP, and the increase could be blocked by anantin. All these results suggest that NPR-A is expressed in bullfrog Müller cells, and activation of the receptor causes an increase of intracellular cGMP levels that activates CNG channels and thereby results in an increased calcium influx.