Chronic administration of indomethacin increases role of nitric oxide in hypercapnic cerebrovasodilation in piglets.

Hypercapnia-induced cerebral vasodilation is associated with prostanoids in the piglet, but is a primarily nitric oxide (NO) associated response in many adult models. Hypercapnia-induced cerebral vasodilation is both NO and prostanoid associated in the juvenile pig. We hypothesized that with chronic administration of indomethacin the piglet would advance the role of the NO system in cerebrovascular responses. The closed cranial window technique was used in piglets to determine pial arteriolar response. Chronically indomethacin treated newborn animals dilated in response to CO2 similarly to control newborns (40.9+/-4.4% vs 48.4+/-4.1%). Topical n-nitro L-arginine (L-NA, 10(-3) M), attenuated CO2 induced dilation in the chronically indomethacin treated animals (11.7+/-3.3% vs 40.9+/-4.4%; p < 0.001), but had no effect on the response to hypercapnia of piglets not treated with indomethacin. Neither indomethacin nor L-NA altered response to topical isoproterenol (10(-6) M). We conclude that with chronic indomethacin administration there develops a significant hypercapnia-induced cerebral vasodilation in which NO has an important role. The chronic inhibition of the newborn's principal dilator system appears to increase the role of NO in newborn cerebral hemodynamics.     

Angiotensin II type 1 (AT1)-receptor blocker prevents impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats

Our aim was to test for smoking-induced endothelial dysfunction in rat cerebral vessels, then to evaluate the effect of valsartan [angiotensin II type I (AT1)-receptor blocker] on that impairment. In pentobarbital-anesthetized, mechanically ventilated Sprague-Dawley rats, we used a cranial window preparation to measure changes in pial vessel diameters following topical applications of acetylcholine (Ach) (before and after smoking or intravenous nicotine infusion; n = 6 in each group), and adenosine (n = 6 for before and after smoking). Then, after intravenous valsartan pretreatment we reexamined the pial vasodilator response to topical Ach (before and after cigarette smoking). Under control conditions, cerebral arterioles were dilated by 6.9 +/- 4.2% and 13.6 +/- 4.8% by topical Ach (10(-6) M and 10(-5) M, respectively) and by 6.4 +/- 2.5% and 12.2 +/- 3.1% by topical adenosine (10(-5) M and 10(-4) M, respectively). One hour after a 1-min inhalation of mainstream smoke (1-mg nicotine cigarette), 10(-5) M Ach constricted cerebral arterioles (-4.4 +/- 4.1%), while 10(-4) M adenosine dilated them by 13.4 +/- 3.4%. One hour after a 1-min nicotine infusion (0.05 mg), 10(-5) M Ach dilated cerebral arterioles by 9.9 +/- 2.4%. Thus, vasodilator response to topical Ach was impaired after smoking, whereas that to adenosine was unaffected. However, the vasodilator response to Ach was unaffected by intravenous nicotine. Valsartan prevented smoking from impairing Ach-induced vasodilation. In conclusion, acute single-cigarette smoking causes a dysfunction of endothelium-dependent, but not endothelium-independent, vasodilation of rat cerebral vessels in vivo, and the effect was not mimicked by intravenous nicotine. AT1-receptor blockade prevented the above smoking-induced impairment of endothelium-dependent vasodilation.     

ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation

This study was designed to test the hypothesis that venular administration of ATP resulted in endothelium-dependent dilation of adjacent arterioles through a mechanism involving cyclooxygenase products. Forty-three male golden hamsters were anesthetized with pentobarbital sodium (60 mg/kg ip), and the cremaster muscle was prepared for in vivo microscopy. ATP (100 microM) injected into venules dilated adjacent arterioles from a mean diameter of 51 +/- 4 to 76 +/- 6 microm (P < 0.05, n = 6). To remove the source of endothelial-derived relaxing factors, the venules were then perfused with air bubbles to disrupt the endothelium. Resting arteriolar diameter was not altered after disruption of the venular endothelium (51 +/- 5 microm), and the responses to venular ATP infusions were significantly attenuated (59 +/- 4 microm, P < 0.05). To determine whether the relaxing factor was a cyclooxygenase product, ATP infusion studies were repeated in the absence and presence of indomethacin (28 microM). Under control conditions, ATP (100 microM) infusion into the venule caused an increase in mean arteriolar diameter from 55 +/- 4 to 78 +/- 3 microm (P < 0.05, n = 6). In the presence of indomethacin, mean resting arteriolar tone was not significantly altered (49 +/- 4 microm), and the response to ATP was significantly attenuated (54 +/- 4 microm, P < 0.05, n = 6). These studies show that increases in venular ATP concentrations stimulate the release of cyclooxygenase products, possibly from the venular endothelium, to vasodilate the adjacent arteriole.     

Transmural gradients of oxygen tension on cerebral microvessels in rats

Using modified oxygen needle microelectrodes, vital microscopy with video-recording facilities, measurements of tissue oxygen tension (PO2) profiles near the cortical arterioles and transmural PO2 gradients on pial arterioles of the rat were performed. At control transmural PO2 gradient averaged 1.17 +/- 0.06 mm Hg/microm (mean +/- SEM, n = 40). Local dilatation of the arteriolar wall (microapplication of sodium nitroprusside approximately 2 x 10(-7) M) resulted in marked drop of the transmural PO2 gradient to 0.68 +/- 0.04 mm Hg/microm (p < 0.001, n = 38). The important finding of the study is the dependence of the transmural PO2 gradient on the vascular tone of pial arterioles. The data presented allow to conclude that O2 consumption of the arteriolar wall lies within the range for surrounding tissue and O2 consumption of the endothelial layer and, apparently, has no substantial impact on transmural PO2 gradient.     

Role of 20-HETE in the pial arteriolar constrictor response to decreased hematocrit after exchange transfusion of cell-free polymeric hemoglobin.

The cerebrovascular response to decreases in hematocrit and viscosity depends on accompanying changes in arterial O2 content. This study examines whether 1) the arteriolar dilation seen after exchange transfusion with a 5% albumin solution can be reduced by the K(ATP) channel antagonist glibenclamide (known to inhibit hypoxic dilation), and 2) the arteriolar constriction seen after exchange transfusion with a cell-free hemoglobin polymer to improve O2-carrying capacity can be blocked by inhibitors of the synthesis or vasoconstrictor actions of 20-HETE. In anesthetized rats, decreasing hematocrit by one-third with albumin exchange transfusion dilated pial arterioles (14 +/- 2%; SD), whereas superfusion of the surface of the brain with 10 muM glibenclamide blocked this response (-10 +/- 7%). Exchange transfusion with polymeric hemoglobin decreased the diameter of pial arterioles by 20 +/- 3% without altering arterial pressure. This constrictor response was attenuated by superfusing the surface of the brain with a 20-HETE antagonist, WIT-002 (10 microM; -5 +/- 1%), and was blocked by two chemically dissimilar selective inhibitors of the synthesis of 20-HETE, DDMS (50 microM; 0 +/- 4%) and HET-0016 (1 microM; +6 +/- 4%). The constrictor response to hemoglobin transfusion was not blocked by an inhibitor of nitric oxide (NO) synthase, and the inhibition of the constrictor response by DDMS was not altered by coadministration of the NO synthase inhibitor. We conclude 1) that activation of K(ATP) channels contributes to pial arteriolar dilation during anemia, whereas 2) constriction to polymeric hemoglobin transfusion at reduced hematocrit represents a regulatory response that limits increased O2 transport and that is mediated by increased formation of 20-HETE, rather than by NO scavenging.     

N-methyl-D-aspartate-induced vasodilation is mediated by endothelium-independent nitric oxide release in piglets

N-methyl-D-aspartate (NMDA) elicits pial arteriolar dilation that has been associated with neuronal nitric oxide (NO) production. However, endothelial factors or glial P-450 epoxygenase products may play a role. We tested whether NMDA-induced pial vasodilation 1) primarily involves NO diffusion from the parenchyma to the surface arterioles, 2) involves intact endothelial function, and 3) involves a miconazole-sensitive component. Arteriolar diameters were determined using closed cranial window-intravital microscopy in anesthetized piglets. NMDA (10-100 microM) elicited virtually identical dose-dependent dilations in paired arterioles (r = 0.94, n = 15). However, NMDA- but not bradykinin (BK)-induced dilations of arteriolar sections over large veins were reduced by 31 +/- 1% (means +/- SE, P < 0.05, n = 4) compared with adjacent sections on the cortical surface. Also, 100 microM NMDA increased cerebrospinal fluid levels of NO metabolites from 3.7 +/- 1.0 to 5.3 +/- 1.2 microM (P < 0.05, n = 6). Endothelial stunning by intracarotid injection of phorbol 12,13-dibutyrate did not affect NMDA-induced vasodilation but attenuated vascular responses to hypercapnia and BK by approximately 70% (n = 7). Finally, miconazole (n = 6, 20 microM) pretreatment and coapplication with NMDA did not alter vascular responses to NMDA. In conclusion, NMDA appears to dilate pial arterioles exclusively through release and diffusion of NO from neurons to the pial surface in piglets.     

Impaired dilation of coronary arterioles during increases in myocardial O(2) consumption with hyperglycemia

Previous studies showed that nitric oxide (NO) plays an important role in coronary arteriolar dilation to increases in myocardial oxygen consumption (MVO(2)). We sought to evaluate coronary microvascular responses to endothelium-dependent and to endothelium-independent vasodilators in an in vivo model. Microvascular diameters were measured using intravital microscopy in 10 normal (N) and 9 hyperglycemic (HG; 1 wk alloxan, 60 mg/kg iv) dogs during suffusion of acetylcholine (1, 10, and 100 microM) or nitroprusside (1, 10, and 100 microM) to test the effects on endothelium-dependent and -independent dilation. During administration of acetylcholine, coronary arteriolar dilation was impaired in HG, but was normal during administration of nitroprusside. To examine a physiologically important vasomotor response, 10 N and 7 HG control, 5 HG and 5 N during superoxide dismutase (SOD), and 5 HG and 4 N after SQ29,548 (SQ; thromboxane A(2)/prostaglandin H(2) receptor antagonist) dogs were studied at three levels of MVO(2): at rest, during dobutamine (DOB; 10 microg. kg(-1). min(-1) iv), and during DOB with rapid atrial pacing (RAP; 280 +/- 10 beats/min). During dobutamine, coronary arterioles dilated similarly in all groups, and the increase in MVO(2) was similar among the groups. However, during the greater metabolic stimulus (DOB+RAP), coronary arterioles in N dilated (36 +/- 4% change from diameter at rest) significantly more than HG (16 +/- 3%, P < 0.05). In HG+SQ and in HG+SOD, coronary arterioles dilated similarly to N, and greater than HG (P < 0.05). MVO(2) during DOB+RAP was similar among groups. Normal dogs treated with SOD and SQ29,548 were not different from untreated N dogs. Thus, in HG dogs, dilation of coronary arterioles is selectively impaired in response to administration of the endothelium-dependent vasodilator acetylcholine and during increases in MVO(2).     

Effects of neuropeptides on growth of cultivated rat molar pulp fibroblasts

The effect of the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) on DNA synthesis of dental pulp cells was investigated in cells grown from molar tooth bud explants from 4–6 days old rat pups. A concentration response-assay of the proliferative response of pulpal cells was performed with SP, NPY, NKA, CGRP and VIP (0.01 to 1 nM) in the presence of EGF (10 ng/ml), hydrocortisone (0.4 μg/ml) and 3% FCS, using [³H]thymidine incorporation. The results showed that SP, NKA and CGRP, but not NPY and VIP, increased the cell number in a concentration-dependent manner, with maxima at 10⁻¹⁰ – 10⁻⁹ M (SP, NKA) and 10⁻⁷ M (CGRP). No potentiating effect was noted when cells are simultaneously stimulated with SP and CGRP. The finding that SP, NKA and CGRP have growth regulatory properties on pulpal cells in vitro suggests that sensory neuropeptides may be involved during pulpal development or in wound healing after pulpal injury.     

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 microg. ml(-1). min(-1) were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10(-7)), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10(-4)-10(-7) M (P < or = 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation (P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.     

Neurokinin A in cerebral vessels: characterization, localization and effects in vitro

Nerve fibres displaying neurokinin A (NKA)-immunoreactivity (IR) were seen in trigeminal nerve cell bodies and around cerebral blood vessels. NKA-positive fibres had the same general distribution as those displaying substance P (SP)-IR. Double or sequential immunostaining revealed coexistence of NKA- and SP-IR in a population of small nerve cell bodies in the trigeminal ganglion and in perivascular nerve fibres of brain vessels; both tachykinins were also noted to coexist with calcitonin gene-related peptide (CGRP)-IR. The presence of NKA- and SP-IR in cerebral vessels from guinea pig was verified by high-performance liquid chromatography and radioimmunochemistry. The levels NKA-IR were higher than those of SP-IR in cerebral vessels of rat, guinea pig and rabbit. In cat, pig, cow and human brain vessels, the levels of NKA- and SP-IR were equal. Major cerebral vessels at the base of the brain contained higher levels of NKA- and SP-IR than pial vessels on the cerebral convexities. Only low levels of NKA-IR and SP-IR were measured in choroid plexus and dura mater. Precontracted isolated arterial segments of middle cerebral (cat), basilar (rabbit, guinea pig and rat) and pial arteries (man) relaxed following the in vitro administration of NKA and SP. The responses occurred in the same concentration range; the IC50 value for NKA was, however, about 10 times higher than that for SP, while the maximum relaxation was equal. In basilar arteries from guinea pig, the peptides NKA, SP and CGRP all induced strong and potent relaxations. There was no evidence that one of the peptides might potentiate the relaxant effects in vitro of another. The present data suggest that NKA, SP and CGRP are costored and can be released together and cooperate in the mediation of vascular reactions in response to activation of the trigemino-cerebrovascular pathway.     

Cortical spreading depression confounds concentration-dependent pial arteriolar dilation during N-methyl-D-aspartate superfusion

Pial arterioles do not express N-methyl-D-aspartate (NMDA) receptors but dilate in response to topical NMDA application. We explored the mechanism underlying NMDA-mediated responses in murine pial arterioles (11-31 microm), using a closed cranial window preparation, and found that arteriolar dilation was not concentration dependent. Pial arteriolar diameter abruptly increased within 3 min of superfusing 50 or 100 microM NMDA. Dilation reached a peak within 1 min (46 +/- 14%) and then declined to a plateau (28 +/- 13%) for the duration of superfusion. Whereas a higher concentration (200 microM) did not produce further dilation, lower concentrations (1-10 microM) did not dilate the arterioles at all. MK-801 (10 microM) abrogated the dilation response, whereas Nomega-nitro-L-arginine (1 mM) attenuated the peak and abolished the sustained dilation during NMDA superfusion. We determined that NMDA-induced pial arteriolar responses were evoked by cortical spreading depression, because abrupt vasodilation during 50 or 100 microM NMDA superfusion was associated with a large negative slow potential shift and electrocorticogram suppression that spread from the superfusion window to distant cortical areas. Our data suggest that the responses of pial arterioles to NMDA are caused in part by neurovascular coupling due to cortical spreading depression.     

Carbon monoxide contributes to hypotension-induced cerebrovascular vasodilation in piglets

The gaseous compound carbon monoxide (CO) has been identified as an important endogenous biological messenger in brain and is a major component in regulation of cerebrovascular circulation in newborns. CO is produced endogenously by catabolism of heme to CO, free iron, and biliverdin during enzymatic degradation of heme by heme oxygenase (HO). The present study was designed to test the hypothesis that endogenously produced CO contributes to hypotension-induced vasodilation of cerebral arterioles. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of the HO substrate heme-l-lysinate caused dilation of pial arterioles that was blocked by a metal porphyrin inhibitor of HO, chromium mesoporphyrin (CrMP). In normotensive piglets (arterial pressure 64 +/- 4 mmHg), CrMP did not cause vasoconstriction of pial arterioles but rather a transient dilation. Hypotension (50% of basal blood pressure) increased cerebral CO production and dilated pial arterioles from 66 +/- 2 to 92 +/- 7 microm. In hypotensive piglets, topical CrMP or intravenous tin protoporphyrin decreased cerebral CO production and produced pial arteriolar constriction to normotensive diameters. In additional experiments, because prostacyclin and nitric oxide (NO) are also key dilators that can contribute to cerebrovascular dilation, we held their levels constant. NO/prostacyclin clamp was accomplished with continuous, simultaneous application of indomethacin, N(omega)-nitro-l-arginine, and minimal dilatory concentrations of iloprost and sodium nitroprusside. With constant NO and prostacyclin, the transient dilator and prolonged constrictor responses to CrMP of normotensive and hypotensive piglets, respectively, were the same as when NO and prostaglandins were not held constant. These data suggest that endogenously produced CO contributes to cerebrovascular dilation in response to reduced perfusion pressure.     

Chronic exposure to nicotine alters endothelium-dependent arteriolar dilatation: effect of superoxide dismutase.

The first goal of this study was to determine whether chronic injection of nicotine alters endothelium-dependent arteriolar dilatation. We measured the diameter of cheek pouch resistance arterioles (approximately 50 microm in diameter) in response to endothelium-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in control hamsters and hamsters treated with nicotine (2 microg. kg-1. day-1 for 2-3 wk). In control hamsters, acetylcholine (0.1 and 1.0 microM) dilated arterioles by 13 +/- 2 and 31 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by 18 +/- 1 and 30 +/- 1%, respectively. In contrast, acetylcholine (0.1 and 1.0 microM) dilated arterioles by only 5 +/- 2 and 12 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by only 7 +/- 2 and 13 +/- 3%, respectively, in animals treated with nicotine (P < 0.05 vs. response in control hamsters). Nitroglycerin produced similar dose-related dilatation of cheek pouch arterioles in control and nicotine-treated hamsters. Our second goal was to examine a possible mechanism for impaired endothelium-dependent arteriolar dilatation during chronic treatment with nicotine. We found that superfusion of the cheek pouch microcirculation with superoxide dismutase (150 U/ml) restored impaired endothelium-dependent, but did not alter endothelium-independent, arteriolar dilatation in hamsters treated with nicotine. Superfusion with superoxide dismutase did not alter endothelium-dependent or -independent arteriolar dilatation in control hamsters. We suggest that chronic exposure to nicotine produces selective impairment of endothelium-dependent arteriolar dilatation via a mechanism related to the synthesis/release of oxygen-derived free radicals.     

Hindlimb unweighting alters endothelium-dependent vasodilation and ecNOS expression in soleus arterioles.

The purpose of this study was to test the hypothesis that endothelium-dependent dilation is impaired in soleus resistance arteries from hindlimb-unweighted (HLU) rats. Male Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 14) or weight-bearing control (Con, n = 14) conditions for 14 days. After the 14-day treatment period, soleus first-order (1A) arterioles were isolated and cannulated with micropipettes to assess vasodilator responses to an endothelium-dependent dilator, ACh (10(-9)-10(-4) M), and an endothelium-independent dilator, sodium nitroprusside (SNP, 10(-9)-10(-4) M). Arterioles from HLU rats were smaller than Con arterioles (maximal passive diameter = 140 +/- 4 and 121 +/- 4 microm in Con and HLU, respectively) but developed similar spontaneous myogenic tone (43 +/- 3 and 45 +/- 3% in Con and HLU, respectively). Arteries from Con and HLU rats dilated in response to increasing doses of ACh, but dilation was impaired in arterioles from HLU rats (P = 0.03), as was maximal dilation to ACh (85 +/- 4 and 65 +/- 4% possible dilation in Con and HLU, respectively). Inhibition of nitric oxide (NO) synthase (NOS) with N(omega)-nitro-L-arginine (300 microM) reduced ACh dilation by approximately 40% in arterioles from Con rats and eliminated dilation in arterioles from HLU rats. The cyclooxygenase inhibitor indomethacin (50 microM) did not significantly alter dilation to ACh in either group. Treatment with N(omega)-nitro-L-arginine + indomethacin eliminated all ACh dilation in Con and HLU rats. Dilation to sodium nitroprusside was not different between groups (P = 0.98). To determine whether HLU decreased expression of endothelial cell NOS (ecNOS), mRNA and protein levels were measured in single arterioles with RT-PCR and immunoblot analysis. The ecNOS mRNA and protein expression was significantly lower in arterioles from HLU rats than in Con arterioles (20 and 65%, respectively). Collectively, these data indicate that HLU impairs ACh dilation in soleus 1A arterioles, in part because of alterations in the NO pathway.     

Mild irritant prevents ethanol-induced gastric mucosal microcirculatory disturbances through actions of calcitonin gene-related peptide and PGI2 in rats

Pretreatment with a mild irritant such as 1 M NaCl prevented ethanol-induced mucosal injury, which was abolished by indomethacin, suggesting involvement of endogenous PGs. With the use of intravital microscopy, we investigated the mechanism in microcirculation whereby a mild irritant prevents ethanol-induced mucosal injury. Microcirculation of the basal part of gastric mucosa in anesthetized rats was observed through a window with transillumination. Diameters of arterioles, collecting venules, and venules were measured with an electric microscaler. One molar NaCl alone caused dilation of arterioles and constrictions of collecting venules and venules, which were inhibited by indomethacin. Ethanol (50%) applied to mucosa constricted collecting venules and venules but dilated arterioles. Constriction of collecting venules resulted in mucosal congestion. Pretreatment with 1 M NaCl inhibited ethanol-induced constrictions of collecting venules and venules, and administration of indomethacin or a calcitonin gene-related peptide (CGRP) antagonist, CGRP-(8-37), abolished elimination of constrictions. Topical application (1 nM-10 microM) of PGE2 or beraprost sodium (a PGI2 analog) to microvasculature markedly and dose-dependently dilated arterioles, whereas that of PGE2, but not beraprost, slightly constricted collecting venules. Pretreatment of microvasculature with a nonvasoactive concentration of PGE2 (100 nM) or beraprost (1 nM) completely inhibited ethanol-induced constriction of collecting venules. The inhibitory effect of beraprost but not of PGE2 was abolished by CGRP-(8-37). Present results suggest that the mechanism whereby 1 M NaCl prevents ethanol-induced injury is elimination of constrictions of collecting venules and venules by CGRP whose release may be enhanced by PGI2 but not by PGE2.     

Governance of arteriolar oscillation by ryanodine receptors

To investigate the role of ryanodine receptors in glomerular arterioles, experiments were performed using an isolated perfused hydronephrotic kidney model. In the first series of studies, BAYK-8644 (300 nM), a calcium agonist, constricted afferent (19.6 +/- 0.6 to 17.6 +/- 0.5 microm, n = 6, P < 0.01) but not efferent arterioles. Furthermore, BAYK-8644 elicited afferent arteriolar oscillatory movements. Subsequent administration of nifedipine (1 microM) inhibited both afferent arteriolar oscillation and constriction by BAYK-8644 (to 19.4 +/- 0.5 microm). In the second group, although BAYK-8644 constricted afferent arterioles treated with 1 microM of thapsigargin (19.7 +/- 0.6 to 16.8 +/- 0.6 microm, n = 5, P < 0.05), it failed to induce rhythmic contraction. Removal of extracellular calcium with EGTA (2 mM) reversed BAYK-8644-induced afferent arteriolar constriction (to 20.0 +/- 0.5 microm). In the third series of investigations, ryanodine (10 microM) but not 2-aminoethoxyphenyl borate (100 microM) abolished afferent arteriolar vasomotion by BAYK-8644. In the fourth series of experiments, in the presence of caffeine (1 mM), the stronger activation of voltage-dependent calcium channels by higher potassium media resulted in greater afferent arteriolar constriction and faster oscillation. Our results indicate that L-type calcium channels are rich in preglomerular but not postglomerular microvessels. Furthermore, the present findings suggest that either prolonged calcium influx through voltage-dependent calcium channels (BAYK-8644) or sensitized ryanodine receptors (caffeine) is required to trigger periodic calcium release through ryanodine receptors in afferent arterioles.     

Evidence that calcitonin gene-related peptide contributes to the capsaicin-induced relaxation of guinea pig cerebral arteries.

Pretreatment with capsaicin caused a depletion of substance P (SP)-, neurokinin A (NKA)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) from the trigeminal ganglion, dura mater and cerebral arteries. The effect of capsaicin on isolated basilar arteries of guinea pig resulted in a biphasic relaxant response of histamine precontracted vessels. The first phase of the capsaicin-induced relaxation was absent in capsaicin-treated guinea pigs. Furthermore, repeated administration of capsaicin decreased the first but not the second phase of relaxation, supporting the view that a stored agent was released, while the second phase probably was due to a direct effect of capsaicin per se. The biphasic effect was not modified by the SP antagonist Spantide in a concentration that blocks tachykinin response (3.10(-6) M), nor by removal of the endothelium. There was no significant difference in pD2 values (-log concentration eliciting half maximum relaxation) between relaxations induced by SP, NKA, neurokinin B, neuropeptide K or CGRP in capsaicin pretreated as compared to vehicle-treated animals. These results are in support of the assumption that CGRP is involved in the capsaicin-induced relaxation caused by release of vasoactive agents from sensory afferent nerves.     

Release of calcitonin gene-related peptide and tachykinins from the rat trachea.

The release of calcitonin gene-related peptide (CGRP), neurokinin A (NKA) and substance P (SP) from intralumenally perfused rat trachea was examined in vitro. In accord with the relative tissue levels of the respective peptides, capsaicin (10(-8) to 10(-5) M) and K+ (120 mM) added to the perfusate resulted in a concentration-dependent increase in the levels of CGRP and NKA, and to a minor extent SP, in the perfusates. Sequential exposure of the trachea to capsaicin revealed a concentration-dependent tachyphylaxis of CGRP release. Thus, 40 min after the application with capsaicin 10(-5) M, a second exposure to capsaicin at the same concentration, or K+ 120 mM, did not evoke CGRP release. In contrast, prior stimulation with K+ 120 mM significantly enhanced the CGRP release induced by a second stimulation with K+ 120 mM or capsaicin 10(-5) M. Capsaicin- and K(+)-induced peptide release was diminished or abolished in the absence of Ca2+. HPLC analysis of CGRP in release materials revealed that there was a single peak which eluted in the same fraction as synthetic rat CGRP. These data demonstrate that CGRP, NKA and SP exist in releasable, capsaicin-sensitive pools in terminals which lie within the proximal lumen of the trachea.     

Arachidonic acid- and prostaglandin E2-induced cerebral vasodilation is mediated by carbon monoxide, independent of reactive oxygen species in piglets

Arachidonic acid (AA) and prostaglandin (PG) E(2) stimulate carbon monoxide (CO) production, and AA metabolism is known to be associated with the generation of reactive oxygen species (ROS). This study was conducted to address the hypothesis that CO and/or ROS mediate cerebrovascular dilation in newborn pigs. Experiments were performed on anesthetized newborn pigs with closed cranial windows. Different concentrations of AA (10(-8)-10(-6) M), PGE(2) (10(-8)-10(-6) M), iloprost (10(-8)-10(-6) M), and their vehicle (artificial cerebrospinal fluid) were given. Piglets with PGE(2) and iloprost received indomethacin (5 mg/kg iv) to inhibit cyclooxygenase. AA, PGE(2), and iloprost caused concentration-dependent increases in pial arteriolar diameter. The effects of both AA and PGE(2) in producing cerebral vascular dilation and associated CO production were blocked by the heme oxygenase inhibitor chromium mesoporphyrin (2 × 10(-5) M), but not by the prostacyclin analog, iloprost. ROS inhibitor tempol (SOD mimetic) (1 × 10(-5) M) and the H(2)O(2) scavenger catalase (1,000 U/ml) also do not block these vasodilator effects of AA and PGE(2). Heme-L-lysinate-induced cerebrovascular dilation and CO production was blocked by chromium mesoporphyrin. Hypoxanthine plus xanthine oxidase, a combination that is known to generate ROS, caused pial arteriolar dilation and CO production that was inhibited by tempol and catalase. These data suggest that AA- and PGE(2)-induced cerebral vascular dilation is mediated by CO, independent of ROS.     

Mechanisms of calcitonin gene-related peptide-induced increases of pulmonary blood flow in fetal sheep

Fetal pulmonary blood flow is regulated by various vasoactive substances. One, calcitonin gene-related peptide (CGRP), increases pulmonary blood flow. We examined four key physiological mechanisms underlying this response using the blocker drugs CGRP receptor blocker (CGRP(8-37)), nitric oxide synthase inhibitor [N(omega)-nitro-L-arginine (L-NNA)], adenosine triphosphate-dependent potassium (K(ATP)) channel blocker (glibenclamide), and cyclooxygenase inhibitor (indomethacin) in 17 near-term fetal sheep. Catheters were placed in the left (LPA) and main pulmonary arteries, and an ultrasonic flow transducer was placed around the LPA to measure flow continuously. CGRP was injected directly into the LPA (mean 1.02 microgram/kg) before and after blockade, and responses to CGRP were statistically compared. Before blockade, CGRP increased LPA blood flow from 23 +/- 25 to 145 +/- 77 ml/min (means +/- SD), and these increases were significantly attenuated by CGRP(8-37) (n = 6; 91% inhibition), L-NNA (n = 6; 86% inhibition), and glibenclamide (n = 6; 69% inhibition). No significant changes were found with indomethacin (n = 6; 4% inhibition). Thus, in the fetal pulmonary circulation, CGRP increases pulmonary blood flow not only through its specific receptor but also, in part, through nitric oxide release and K(ATP) channel activation.